KR20080106919A - Improved Prodrugs of CC-1065 Analogs - Google Patents

Abstract

Described herein is a prodrug of an analog of the antitumor antibiotic CC-1065 having a cleavable protecting group containing sulfonic acid containing phenyl carbamate, which gives the prodrug enhanced water solubility, Drugs have residues, such as sulfides or disulfides, that can be conjugated with cell binding reagents such as antibodies. Therapeutic uses of such prodrug conjugates are also described, prodrugs of such cytotoxic agents are intended for therapeutic use because they deliver cytotoxic prodrugs to specific cell populations and are enzymatically converted to cytotoxic drugs in a targeted manner. I have it.

Description

Improved prodrugs of CC-1065 analogs

The present invention relates to novel prodrugs and therapeutic uses thereof of cytotoxic agents. More specifically, the present invention relates to novel prodrugs of cytotoxic agents, which are analogs of CC-1065 and contain both residues for chemical linkage with cell binding agents and protecting groups cleaved in vivo. Such prodrugs can be chemically linked with cell binding agents to provide therapeutic agents that can be activated and released in vivo and delivered in a targeted manner to a particular cell population.

Many reports have been published on targeting tumor cells using monoclonal antibody-drug conjugates. Sela et al, in Immunoconjugates , pp. 189-216 (C. Vogel, ed. 1987); Ghose et al, in Targeted Drugs , pp. 1-22 (E. Goldberg, ed. 1983); Diener et al, in Antibody Mediated Delivery Systems , pp. 1-23 (J. Rodwell, ed. 1988); Pietersz et al, in Antibody Mediated Delivery Systems , pp. 25-53 (J. Rodwell, ed. 1988); Bumol et al, in Antibody Mediated Delivery Systems , pp. 55-79 (J. Rodwell, ed. 1988); GA Pietersz & K. Krarau, 2 J. Drug Targeting , 183-215 (1994); RVJ Chari, 31 Adv. Drug Delivery Revs., 89-104 (1998); WA Blattler & RVJ Chari, in Anticancer Agents , Frontiers in Cancer Chemotherapy , 317-338, ACS Symposium Series 796; and I. Ojima et al eds, American Chemical Society 2001]. Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, and maytansinoids can be applied to various murine monoclonal antibodies. It has been bonded. In some cases, the drug molecule is described as serum albumin [Ganett et al, 46 Cancer Res . 2407-2412 (1986); Ohkawa et al, 23 Cancer Immunol. Immunother . 81-86 (1986); Endo et al, 47 Cancer Res . 1076-1080 (1980)], dextran [Hurwitz et al, 2 Appl. Biochem . 25-35 (1980); Manabi et al, 34 Biochem. Pharmacol . 289-291 (1985); Dillman et al, 46 Cancer Res . 4886-4891 (1986); and Shoval et al, 85 Proc. Natl. Acad. Sci. USA 8276-8280 (1988)], or polyglutamic acid (Tsukada et al, 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al, 27 J. Med. Chem . 1602-1607 (1984); Tsukada et al, 52 Br. J. Cancer 111-116 (1985) to an antibody molecule via an intermediate carrier molecule.

A wide range of linker arrays for making such immunoconjugates are currently available, including both cleavable and non-cleavable linkers. However, in vitro cytotoxicity tests have shown that antibody-drug conjugates hardly exert the same cytotoxic effects as unconjugated free drugs. This suggests that the mechanism by which drug molecules are released from conjugated antibodies is extremely inefficient. Initial research in the field of immunotoxins revealed that conjugates formed through disulfide bridges between monoclonal antibodies and catalytically active protein toxins are more cytotoxic than conjugates containing other linkers. Lambert et al, 260 J. Biol. Chem . 12035-12041 (1985); Lambert et al, in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie et al, 48 Cancer Res . 2610-2617 (1988). This improved cytotoxicity was due to the presence of high concentrations of reduced glutathione in the cells, which helped to efficiently cleave the disulfide bonds between the antibody molecule and the toxin. Maytansinoids and calicheamicin are the first examples of highly cytotoxic drugs that link monoclonal antibodies via disulfide bonds. The antibody conjugates of these drugs have been shown to have potent potency in vitro and to have excellent antitumor activity in the human tumor xenograft model of mice. RVJ Chari et al., 52 Cancer Res ., 127 -131 (1992); C. Liu et al., 93, Proc. Natl. Acad. Sci. 8618-8623 (1996); LM Hinman et al., 53, Cancer Res ., 3536-3542 (1993); and PR Hamann et al, 13, BioConjugate Chem ., 40-46 (2002).

A candidate of interest in preparing such cytotoxic conjugates is CC-1065, which is a Streptomyces zelensis ) is a potent antitumor antibiotic isolated from culture. CC-1065 is about 1000 times more potent in vitro than commonly used anticancer drugs such as doxorubicin, methotrexate and vincristine [BK Bhuyan et al., Cancer Res ., 42, 3532-3537 (1982). The structure of CC-1065 (Compound 1, FIG. 1A ) was determined by x-ray crystallography [Martin, DG et al, 33 J. Antibiotics 902-903 (1980), and Chidester, CG, et al, 103 J. Am. Chem. Soc . 7629-7635 (1981). The CC-1065 molecule consists of three substituted pyrroloindole residues linked by amide bonds. “A” subunits have a cyclopropyl ring containing only one asymmetric carbon in the molecule. Only the relative steric configuration of these carbons can be obtained from x-ray data, but the absolute steric configuration was inferred as 3b- R , 4a- S by using DNA as a chiral reagent. Hurley, LH et al, 226 Science 843- 844 (1984). "B" and "C" subunits of CC-1065 are the same pyrroloindole residue.

The cytotoxic potency of CC-1065 has been correlated with its alkylation activity and its DNA-binding activity or DNA-insertion activity. These two activities are in separate parts of the molecule. That is, alkylation activity is contained in cyclopropyrroloindole (CPI) subunits and DNA-binding activity is present in two pyrroloindole subunits ( FIG. 1A ).

However, although CC-1065 has certain characteristics that are of interest as cytotoxic agents, there are limits to their therapeutic use. Administration of CC-1065 to mice induced delayed hepatotoxicity, which resulted in death 50 days after 12.5 μg / kg intravenous administration [VL Reynolds et al., J. Antibiotics , XXIX, 319- 334 (1986)]. This has spurred efforts to develop analogs that do not cause delayed toxicity, and the synthesis of simpler analogs modeled after CC-1065 has been reported. MA Warpehoski et al., J. Med. Chem. , 31, 590-603 (1988). In another series of analogues, CPI residues were replaced with cyclopropabenzindole (CBI) residues. See DL Boger et al., J. Org. Chem ., 55, 5823-5833, (1990), DL Boger et al., BioOrg. Med. Chem. Lett. , 1, 115-120 (1991). These compounds maintain the potent in vitro potency of the parent drug without causing delayed toxicity in mice. Like CC-1065, these compounds are alkylating agents that bind to the minor grooves of DNA in a covalent manner, causing cell death. However, the clinical evaluation of the most promising analogs Adozelesin and Carzelesin was disappointing [BF Foster et al., Investigational New Drugs , 13, 321-326 (1996). ); I. Wolff et al., Clin. Cancer Res. , 2, 1717-1723 (1996). These drugs exhibited insufficient therapeutic effects because of their high systemic toxicity.

The therapeutic efficacy of the CC-1065 analog can be significantly improved by altering the distribution in vivo through targeted delivery to the tumor site, thereby further reducing toxicity to non-targeted tissue, thereby lowering systemic toxicity. To achieve this goal, conjugates of cell binders that specifically target tumor cells with analogs and derivatives of CC-1065 have been reported. See US Pat. No. 5,475,092; No. 5,585,499; 5,846,545]. These conjugates typically exhibited high target-specific cytotoxicity in vitro and excellent antitumor activity in human tumor xenograft models of mice. See RVJ Chari et al., Cancer Res ., 55, 4079-4084 (1995)].

Cell binders are typically soluble only in aqueous media and are usually stored in aqueous solution. Therefore, these analogs should have sufficient water solubility to allow efficient reaction with the cell binder and subsequent formulation in aqueous solution. In addition, for cell binder conjugates to have a useful shelf life, it is important that CC-1065 analogs linked with these cell binders are stable for a long time in aqueous solution.

The CC-1065 analogs reported so far [see, eg, FIGS. 1B and 1C ] are almost water insoluble. Due to the insolubility of the CC-1065 analogues, conjugation reactions with current cell binders must be performed in extremely dilute aqueous solutions. Therefore, these prodrugs should have enhanced water solubility compared to the parent drug.

In addition, the CC-1065 analogs reported so far are somewhat unstable in aqueous solutions for the following reasons. The seco -form of the drug may be spontaneously converted to the cyclopropyl form and then alkylated if present. However, the competitive reaction of the cyclopropyl form with water results in the ring opening of the cyclopropyl ring resulting in an inert hydroxy compound. Thus, for example, by developing prodrugs of CC-1065 analogs, it is necessary to protect the reactive portion of the CC-1065 analogs in order to extend their useful life in aqueous solutions.

Thus, there is a need to develop prodrugs of CC-1065 analogs that are extremely stable upon storage in aqueous solution. Preferably these prodrugs should be converted only to the active drug in vivo. Once such a prodrug is injected into a patient, it should preferably be efficiently converted to the active drug.

Carzelesin is a prodrug that protects phenolic groups in adozelesin as phenyl carbamate. LH Li et al., Cancer Res. , 52, 4904-4913 (1992). However, these prodrugs are too unstable for therapeutic use and provide no increase in water solubility compared to the parent drug. In the second example, the phenolic residues of the CC-1065 analog were glycosylated to produce prodrugs (US Pat. No. 5,646,298). However, these prodrugs have not been converted into active drugs in vivo and require additional administration of enzymes from bacterial sources to convert them into cytotoxic forms.

There are several examples of anticancer drugs that have been converted to water-soluble prodrugs but are not related to CC-1065. In the case of the anticancer drug irinotecan, the phenolic group is protected by 4-piperidino-piperidino carbamate. Such protecting groups have been reported to impart water solubility to the drug. In addition, the prodrugs are readily converted into active drugs in humans in vivo, presumably due to the enzyme carboxyl esterase that is naturally present in human serum, tumor tissue and some organs. See A. Sparreboom. , 4, Clin. Cancer Res ., 2747-2754 (1998). LP Rivory et al., 52, Biochem Pharmacol ., 1103-1111 (1996).

Similarly, the anticancer drug etoposide phosphate is an example of a prodrug with a phosphate protecting group that is rapidly converted into the active drug in vivo, possibly through hydrolysis by an endogenous alkaline phosphatase [SZ Fields et al., 1 Clin. Cancer Res ., 105-111 (1995).

Recently, R.Y. Zhao et al. (US 6,756,397 B2) describe water-soluble prodrugs of the first class of CC-1065 analogues comprising carbamate or phosphate substituents on the phenolic ring of the alkylated portion of the molecule. We have found that the solubility of compounds containing these types of substituents may not be satisfactory under physiological conditions. In addition, specific agents such as phosphatase must act in order to convert these compounds into biologically active forms. Thus, there is a general need for analogs of CC-1065 that can increase solubility in aqueous solutions and / or readily dissolve even under physiological conditions. In addition, it is highly desirable to promote conjugation with cell binders in aqueous solution while maintaining their biological activity. In addition, to reduce toxic side effects, it would be advantageous to provide CC-1065 analogs in the form of prodrugs, which are converted primarily to cytotoxic drugs, preferably through spontaneous hydrolysis at desired site of treatment. All of these advantages are provided by the invention described herein, which will be apparent to one skilled in the art from the following detailed description and examples.

Summary of the Invention

It is an object of the present invention to provide prodrugs of CC-1065 analogs which have enhanced solubility in aqueous media. These and other objects have been achieved by providing prodrugs in which the phenolic group of the alkylated portion of the molecule is protected with a functional group that makes the drug stable upon storage in acidic aqueous solution. In addition, such protecting groups increase the water solubility of the drug compared to unprotected analogs. Protective groups are readily cleaved in vivo under physiological pH to provide the corresponding active drug. In the prodrugs described herein, the water solubility was enhanced by protecting the phenolic substituents as sulfonic acid containing phenyl carbamate, which retains charge under physiological pH. To further enhance water solubility, any polyethylene glycol spacer can be introduced into the linker between the cleavable linkage, such as a disulfide group, and the indolyl subunit. Introduction of such spacers does not alter the potency of the drug.

More specific embodiments of the present invention provide prodrugs comprising analogs of seco -cyclopropagenzindole-containing cytotoxic drugs that have protecting groups that enhance water solubility and stability and can be cleaved in vivo under physiological pH. do. The prodrug of this specific embodiment has a first subunit and a second subunit linked by an amide bond from the secondary amino group of the pyrrole residue of the first subunit to the C-2 carboxyl of the second subunit. Such a first subunit is shown as a compound of Formula I, which is a compound of Formulas II to XI, or a pharmaceutically acceptable salt, hydrate or hydrate salt thereof, or a polymorphic crystalline structure of these compounds or an optical isomer thereof Conjugated with a second subunit selected from semi-, diastereomers or enantiomers:

Where

Y represents a leaving group selected from halides (fluoride, chloride, bromide or iodide), methanesulfonate, para-toluenesulfonate, trifluoromethane sulfonate, mononitro or dinitrophenolate. Preferably, the leaving group is chloride or bromide.

R, R ', R ₁ -R ₆ are each independently hydrogen, C ₁ -C ₃ linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino or amido, or a cell binder And a linker that provides a linkage to a prodrug, the linkage being preferably made via a sulfide or disulfide bond, provided that at least one of R, R ', R ₁ -R ₆ is a linker. Preferably, R is a linker. The linker may comprise a polyethylene glycol spacer.

R ₇ is a protecting group that can be cleaved in vivo and can enhance the water solubility of cyclopropabenzindole-containing cytotoxic drugs and is sulfonic acid containing phenyl carbamate. R ₇ has the formula

[Wherein R ₉ is H or C ₁ -C ₄ branched or linear alkyl; R _10, R _11, R ₁₂ or R ₁₃ are the same or different and are one or more double -X-SO ₃ ^- groups inde ⁺ M, M ⁺ is a cation derived from the atoms of the H ⁺ or a Group IA, e. For example Na ⁺ , K ^+, etc., X is a direct bond or a spacer selected from C1 to C4 linear or branched alkyl, alkenyl or alkynyl, -Oalkyl-, -Salkyl- and aminoalkyl, and other R ₁₀ , R ₁₁ , R ₁₂ or R ₁₃ is H, C ₁ -C ₆ branched or linear alkyl, —Oalkyl, —Salkyl, hydroxyl, primary amino, secondary amino, or amido, halide, nitro, azido ].

According to a preferred embodiment, the prodrugs of the invention are compounds of the formula: or pharmaceutically acceptable salts, hydrates or hydrates thereof, or polymorphic crystalline structures of these compounds or optical isomers, racemates, diastereomers thereof or It is an enantiomer:

Where

Y, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R are as defined above and R is a linker as defined herein.

Preferably, the prodrug is selected from the group consisting of:

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-methyldithiopropanoyl) -amino] -1H-indol-2-carboxamide;

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide;

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-{[3- (2-pyridyldithio) -propanoyl] -amino} -1H-indol-2-carboxamide;

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide;

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide; or

Pharmaceutically acceptable salts, hydrates or hydrated salts thereof, or polymorphic crystalline structures of these compounds or optical isomers, racemates, diastereomers or enantiomers thereof.

According to another preferred embodiment, the prodrugs of the present invention are compounds of the formula: or pharmaceutically acceptable salts, hydrates or hydrates thereof, or polymorphic crystalline structures of these compounds or optical isomers, racemates, diasteres thereof Isomers or ethiomers are:

In the above formula,

Y, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R are as defined above and R is a linker as defined herein.

Preferably, the prodrug is selected from the group consisting of:

(S) -3-((1- (chloromethyl) -3- (5- (3-mercaptopropaneamido) -1H-indole-2-carbonyl) -2,3-dihydro-1H-benzo [e] indol-5-yloxy) carbonylamino) benzenesulfonic acid;

(S) -3-((1- (chloromethyl) -3- (5- (3- (methyldisulfanyl) propaneamido) -1H-indole-2-carbonyl) -2,3-dihydro -1H-benzo [e] indol-5-yloxy) carbonylamino) benzenesulfonic acid; or

Pharmaceutically acceptable salts, hydrates or hydrated salts thereof, or polymorphic crystalline structures of these compounds or optical isomers, racemates, diastereomers or enantiomers thereof.

Prodrugs of the invention can be used in cytotoxic conjugates in which cell binding agents are linked to one or more prodrugs of the invention via a linking group.

The linking group includes a linker as defined above linked to a functional group reactive to thiol, sulfide or disulfide of the cell binder.

Cell binding agents include antibodies and fragments thereof, interferons, lymphokines, vitamins, hormones and growth factors. Pharmaceutical compositions containing such conjugates are also provided.

Cytotoxic conjugates can be used in methods of treating a particular subject by administering an effective amount of the pharmaceutical composition. Depending on the cell type that binds the selected cell binding agent, many diseases can be treated in vivo, ex vivo or in vitro. Such diseases include, for example, many types of cancers, including lymphomas, leukemias, lung cancers, breast cancers, colon cancers, prostate cancers, kidney cancers, pancreatic cancers, and the like.

Thus, CC-1065 is useful for improving solubility and stability in aqueous solutions, retaining cytotoxicity when activated, producing alkylated drugs, and targeting specific cell types through conjugation with specific cell binding agents. Prodrugs of the analogs are provided.

1A shows the structure of CC-1065 and its subunits A, B, and C. FIG.

1B and 1C show the structure of two known analogs of CC-1065.

1D depicts the preparation of intermediate DC1SMe (see US Pat. No. 6534660 B1).

2 illustrates the structure of the CC-1065 analogs and prodrugs of the present invention which are illustrated.

Figure 3 illustrates the structure of the polyethylene glycol-containing prodrug of the invention illustrated.

4A and C show (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylphenyl) aminocarbonyloxy] -3H-benz (e) indol-3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-methyldithiopropanoyl) -amino] -1 H-indole-2-carboxamide (DC07SMe ); (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide (DC07); (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-{[3- (2-pyridyldithio) -propanoyl] -amino} -1H-indole-2-carboxamide ( DC08SPy); And (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole -3-yl} carbonyl] -1 H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1 H-indol-2-carboxamide (DC08); (S) -3-((1- (chloromethyl) -3- (5- (3- (methyldisulfanyl) propaneamido) -1H-indole-2-carbonyl) -2,3-dihydro -1H-benzo [e] indol-5-yloxy) carbonylamino) benzenesulfonic acid (DC101SMe); (S) -3-((1- (chloromethyl) -3- (5- (3-mercaptopropaneamido) -1H-indole-2-carbonyl) -2,3-dihydro-1H-benzo [e] Synthesis scheme for preparing indol-5-yloxy) carbonylamino) benzenesulfonic acid (DC107).

5 is a schematic of the activation / deactivation process of a drug under physiological pH.

FIG. 6 shows in vitro cytotoxicity data of DC07-SMe and DC1-SMe after 5 days of exposure to Ramos and HL60 cells.

7 depicts in vitro cytotoxicity data of DC1-SPy and DC07-Spy for Ramos and HL60 cells.

8 depicts in vitro cytotoxicity data of DC08-Spy against Ramos and Namalwa cells.

9 depicts in vitro cytotoxicity data of DC101-SMe and DC107-SMe against HL-60 and Ramos cells.

10 depicts in vitro cytotoxicity data of MY9-6-DC07 (disulfide linked) for KARA and Ramos cells.

FIG. 11 depicts in vitro cytotoxicity data of huC242-DC07 (thioether linked) for COLO205 & A-375 cells.

We have found that the stability, water solubility and utility of certain CC-1065 analogs are enhanced by protecting the alkylated moieties of such analogs with suitable protecting groups. This provided us with a prodrug of CC-1065 analogues that enhanced water solubility and could additionally be linked to cell binding agents, whereby the therapeutic efficacy of the prodrugs of such CC-1065 analogs was targeted to the tumor site. Delivery improves by changing the distribution in vivo to lower toxicity to non-targeted tissue, thereby lowering systemic toxicity. Physiological pH substantially converts the prodrug into its active drug form, and in embodiments with a cleavable linker for cell binders, the active drug form of the CC-1065 analog is released, thereby further enhancing its cytotoxic activity. On the other hand, the linker to the cell binding agent may be first cleaved inside the target cell to release the prodrug, after which it is endogenously converted to the active drug.

To achieve this goal, the inventors have found that (a) the first subunit of formula (I) wherein phenolic hydroxyl is protected by a protecting group and cleaved in vivo under physiological pH to enhance water solubility, and (b) CC-, which is a seco -cyclopropagenzindole (CBI) -containing cytotoxic prodrug having a structure shown as one of II-XI and comprising a second subunit comprising a linker for conjugating the prodrug to the cell binder. Exemplary prodrugs of the 1065 analogs ( FIGS. 2-4B ) were synthesized. The linker may contain polyethylene glycol spacers ( FIG. 3 ). Removal of the prodrug's protecting group produces an active form of the drug that retains the high cytotoxicity of the parent drug. Linkers are used for conjugation with cell binders, preferably comprising disulfide bonds or sulfide (or thioethers) bonds herein.

Linking highly cytotoxic drugs to antibodies using cleavable linkages, such as disulfide bonds, results in the release of fully active drugs inside the cell and that these conjugates are cytotoxic in an antigen-specific manner. [Reference: RVJ Chari et al, 52 Cancer Res. 127-131 (1992); R.V.J. Chari et al., 55 Cancer Res. 4079-4084 (1995); And US Pat. Nos. 5,208,020 and 5,475,092. In the present invention, we describe the synthesis of prodrugs of CC-1065 analogs, the conjugation of them with monoclonal antibodies, and the procedure for evaluating the in vitro cytotoxicity and specificity of such conjugates. Thus, the present invention is directed to killing or lysing diseased or abnormal cells, such as tumor cells, virus infected cells, microbial infected cells, parasitic infected cells, autoimmune cells (cells that produce auto-antibodies) with minimal side effects. ), Compounds that are useful for preparing a therapeutic agent intended to remove activated cells (cells involved in graft rejection or graft versus host disease), or any other type of diseased or abnormal cell.

Thus, the present invention teaches the synthesis of prodrug analogs and derivatives of CC-1065 that can be chemically linked to cell binding agents that maintain high cytotoxicity of parent compound CC-1065 upon release of a protecting group. In addition, when these compounds are linked to cell binding agents upon activation, they are cytotoxic to cells that bind cell binding agents and much less toxic to non-target cells.

Prodrug of the Invention

Prodrugs according to the present invention comprise a CC-1065 analogue that protects the phenolic group of the alkylated portion of the molecule, and the prodrugs of the present invention further comprise a linker capable of conjugating it with a cell binder. The prodrug may comprise first and second subunits linked via amide bonds.

According to a particular embodiment of the invention, the prodrug of the CC-1065 analog has a first subunit which is seco- CBI (cyclopropabenzindole unit) in its open halomethyl form, which first subunit is in vivo It has a phenolic hydroxyl protected by a water-soluble protecting group that can be cleaved at The second subunit of a prodrug of certain embodiments of the present invention is a B-subunit or B subunit and a C subunit of CC-1065, which are either 2-carboxy-indole or 2-carboxy-benzofuran derivatives or both ( FIG. 1 ). Analogs of combinations, which are represented by Formulas II-XI. As can be seen from the properties of native CC-1065 and published published analogues, see, eg, Warpehoski et al, 31 J. Med. Chem. 590-603 (1988), Boger et al, 66 J. Org. Chem. 6654-6661 (2001)], B and C subunits may also contain other heterocycles or substituted heterocycles, and thus different on indole or benzofuran rings, corresponding to positions R ₁ to R ₆ of Formulas II to XI. It may involve different substituents at the position and still retain potent cytotoxic activity.

In order to link prodrugs of CC-1065 analogs with cell binders, the prodrugs must first be disulfide bonds, sulfide (or referred to herein as thioethers) bonds, acid-labile groups, photo-labile groups, peptidase- It should include residues that allow linkage with the cell binding agent through linkages such as labile groups, or esterase-labile groups. The prodrug analogs contain, for example, the residues needed to bind the cell binder via disulfide bonds, thioether bonds, acid-labile groups, photolabile groups, peptidase-labile groups, or esterase-labile groups. Prodrug analogs are prepared. To further enhance solubility in aqueous solution, the linker may contain polyethylene glycol spacers ( FIG. 3 ).

Preferably a disulfide linkage is used, which is due to the reducing environment of the targeting cell to which the disulfide is cleaved and the prodrug (or drug, according to the relative order of cleavage of the prodrug from the cell binder and hydrolysis of the protecting group) is released. In this case, the cytotoxicity is increased.

More specifically, in accordance with certain embodiments of the present invention, the prodrug of the CC-1065 analog is from the secondary amino group of the pyrrole moiety of the first subunit to the C-2 carboxy group of the second subunit having Formulas II to XI. It includes the first subunit and the second subunit covalently linked via an amide bond of.

Linkers in Formulas II-XI allow prodrugs of the CC-1065 analogs to be linked to cell binders. Such linkers may contain polyethylene glycol spacers. Examples thereof include residues that can be linked through disulfide bonds, thioether bonds, acid-labile groups, photolabile groups, peptidase-labile groups, or esterase-labile groups, and are well known in the art. [See, eg, US Pat. No. 5,846,545; Which is incorporated herein by reference]. Preferred residues are residues which can be linked via disulfide bonds, for example thiols (DC1, DC07, DC08) or disulfides (DC1-SMe, DC07-SMe, DC08SPy, see FIGS. 2-4B ). Mixed disulfides containing terminal leaving groups such as thiomethyl (DC1-SMe, DC07-SMe), DCO8SPy, glutathione, alkyl thiols, pyridylthio, aryl thiols, nitropyridylthio, hydroxycarbonylpyridylthio, (Nitro) hydroxycarbonyl-pyridylthio and the like can be used provided that such disulfides are capable of undergoing a disulfide-exchange reaction for coupling the prodrug with the cell binder. The linker may optionally further comprise a spacer region interposed between the reactive group of the moiety that enables linkage and the reactive group of the 2-carboxy-indole or 2-carboxy-benzofuran derivative moiety.

Preferably, the linker has the formula:

-G-D- (Z) p-S-Z '

In the above formula,

G is a single or double bond, -O-, -S- or -NT-;

D is a single bond or -E-, -E-NT-, -E-NT-F-, -EO-, -EOF-, -E-NT-CO-, -E-NT-CO-F-,- E-CO-, -CO-E-, -E-CO-F, -ES-, -ESF-, -E-NT-CS-, -E-NT-CS-F-;

T is H or C ₁ -C ₆ branched or linear alkyl;

E and F are the same or different and are linear or branched-(OCH ₂ CH ₂ ) _i alkyl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i -alkyl-,-(OCH ₂ CH ₂ ) _i -,-(OCH ₂ CH ₂ ) _i Cycloalkyl (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i Heterocyclic (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i Aryl (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i heteroaryl (OCH ₂ CH ₂ ) _j- , -alkyl- (OCH ₂ CH ₂ ) _i alkyl (OCH ₂ CH ₂ ) _j -,- Alkyl (OCH ₂ CH ₂ ) _i- , -alkyl- (OCH ₂ CH ₂ ) _i Cycloalkyl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i Heterocyclic (OCH ₂ CH ₂ ) _j -, -Alkyl (OCH ₂ CH ₂ ) _i aryl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i heteroaryl (OCH ₂ CH ₂ ) _j- , cycloalkyl-alkyl-, -alkyl- Independently selected from cycloalkyl-, -heterocyclic-alkyl-, -alkyl-heterocyclic-, -alkyl-aryl-, aryl-alkyl-, -alkyl-heteroaryl- and -heteroaryl-alkyl-;

i and j are the same or different integers and are independently selected from 0, 1 to 2000;

Z is linear or branched-alkyl-;

p is 0 or 1;

Z 'is H, a thiol protecting group, for example COR, R ₂₀ or SR _2O , wherein R ₂₀ represents H, methyl, alkyl, optionally substituted cycloalkyl, aryl, heteroaryl or heterocyclic.

The following aspects or all combinations thereof are preferred:

G is a single bond or -NT-;

G is -NT-;

D is -CO-E-;

E is linear or branched-alkyl-;

p is 0;

Z 'is H or SR _2O and R ₂₀ represents alkyl or heteroaryl.

Preferred embodiments of the linker include (CH ₂ ) _p NHCO (CH ₂ ) _m SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m SZ, (CH ₂ ) _p O (CH ₂ ) _m SZ, (CH ₂₎ ) _p NHCO (CH ₂ ) _m (OCH ₂ CH ₂ ) _n SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n SZ, (CH ₂ ) _p O (CH ₂ ) _m (OCH ₂ CH ₂ ) _n SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p O (CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CH (Me) SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CH (Me) SZ, (CH ₂ ) _p O (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CH (Me) SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m C (Me) ₂ SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m C (Me) ₂ SZ, (CH ₂ ) _p O (CH ₂ ) _m C (Me) ₂ SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m (OCH ₂ CH ₂ ) _n C (Me) ₂ SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n C (Me) ₂ SZ, ( CH ₂ ) _p O (CH ₂ ) _m (OCH ₂ CH ₂ ) _n C (Me ₂ ) SZ, (CH ₂ ) _p OC ₆ H ₄ (CH ₂ ) _m SZ, (CH ₂ ) _p OC ₆ H ₄ ( CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p OC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CHMeSZ, (CH ₂ ) _p OC ₆ H ₄ (CH ₂ ) _m CMe ₂ SZ Or (CH ₂ ) _p OC ₆ H ₄ (CH ₂ ) _m (OC H ₂ CH ₂ ) _n C (Me) ₂ SZ [where Z represents H, a thiol protecting group such as Ac, R ₈ or SR ₈ and R ₈ represents methyl, linear alkyl, branched alkyl, cyclic alkyl , A simple or substituted aryl or heterocyclic, m represents an integer from 0 to 10, n represents an integer from 0 to 2000, p represents an integer from 0 to 10.

Examples of linear alkyl represented by R ₈ include methyl, ethyl, propyl, butyl, pentyl and hexyl. Examples of branched alkyl represented by R ₈ include isopropyl, isobutyl, secondary-butyl, tert-butyl, isopentyl and 1-ethyl-propyl. Examples of cyclic alkyl represented by R ₈ include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Examples of simple aryls represented as R ₈ include phenyl and naphthyl. Examples of substituted aryls represented by R ₈ include alkyl groups, halogens such as phenyl or naphthyl substituted with Cl, Br, F, nitro groups, amino groups, sulfonic acid groups, carboxylic acid groups, hydroxy groups and alkoxy groups; The same aryl is included. Heterocyclics represented by R ₈ are compounds in which the hetero atom is selected from O, N, and S, examples of which include furyl, pyrrolyl, pyridyl (eg, 2-substituted pyrimidine groups) and thiophene.

Most preferred embodiments of the linker include (CH ₂ ) _p NHCO (CH ₂ ) ₂ SH, (CH ₂ ) _p NHCO (CH ₂ ) ₂ C (Me) ₂ SH, (CH ₂ ) _p NHCO (CH ₂ ) ₂ SSCH ₃ , (CH ₂ ) _p NHCO (CH ₂ ) ₂ (OCH ₂ CH ₂ ) _n SH, (CH ₂ ) _p NHCO (CH ₂ ) ₂ (OCH ₂ CH ₂ ) _n C (Me) ₂ SSCH ₃ and (CH ₂ ) _p NHCO (CH ₂ ) ₂ (OCH ₂ CH ₂ ) _n SSMe.

Within Formulas II-XI, R, R ', R _1- R ₆ may be the same or different and independently represent hydrogen, C ₁ -C ₃ linear alkyl, methoxy, hydroxyl, primary amino, secondary amino , Tertiary amino or amido, or a linker, provided that at least one of R, R ', R ₁ to R ₆ is a linker. Preferably R is a linker. Examples of primary amino group-containing substituents are methyl amino, ethyl amino and isopropyl amino. Examples of secondary amino group-containing substituents are dimethyl amino, diethyl amino and ethyl-propyl amino. Examples of amino groups include N-methyl-acetamido, N-methyl-propionamido, N-acetamido and N-propionamido.

Within Formulas II-XI, R ₇ is an in vivo-cleavable protecting group that enhances the water solubility of seco -cyclopropagenzindole-containing cytotoxic drugs, such groups being sulfoic acid containing phenyl carbamate of Formula XII Can be chosen from:

Formula XII

In the above formula,

R ₉ is H or C ₁ -C ₄ branched or linear alkyl; R _10, R _11, R ₁₂ or R ₁₃ are the same or different and are one or more double -X-SO ₃ ^- groups inde ⁺ M, M ⁺ is a cation, for, for example, derived from the atom of the H ⁺ or a Group IA Na ⁺ , K ⁺ and the like, X is a direct bond or a spacer selected from C1 to C4 linear or branched alkyl, alkenyl or alkynyl, -Oalkyl-, -Salkyl- and aminoalkyl, other R ₁₀ , R ₁₁ , R ₁₂ or R ₁₃ is H, C ₁ -C ₆ branched or linear alkyl, —Oalkyl, —Salkyl, hydroxyl, primary amino, secondary amino, or amido, halide, nitro, azido.

Examples of linear alkyl represented by R ₉ , R ₁₀ , R ₁₁ , R ₁₂ or R ₁₃ include methyl, ethyl, propyl, butyl, pentyl and hexyl. Examples of branched alkyl represented by R ₉ , R ₁₀ , R ₁₁ , R ₁₂ or R ₁₃ include isopropyl, isobutyl, secondary-butyl, tert-butyl, isopentyl and 1-ethyl-propyl. Examples of cyclic alkyl represented by R ₉ , R ₁₀ , R ₁₁ , R ₁₂ or R ₁₃ include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Thus, sulfonic acid containing phenyl carbamate has been found to be cleavable by hydrolysis under physiological pH that occurs in serum and plasma.

The sulfide or disulfide-containing and mercapto-containing prodrugs of the CC-1065 analogs of the present invention can be evaluated for their ability to inhibit the proliferation of unnecessary various cell lines in vitro under incubated conditions. For example, cell lines such as Ramos cell lines and HL60 can be readily used to assess the cytotoxicity of these compounds. The cells to be assessed can be exposed to the compound for 24 hours and the viable cell fractions measured by direct assay by known methods. The IC ₅₀ value can then be calculated from these assay results.

As used herein, the expression “connectable with a cell binder” refers to a CC-1065 analog prodrug comprising one or more linkers or precursors thereof suitable for binding the derivative to the cell binder; Preferred linkers contain thiols, sulfide or disulfide bonds, or precursors thereof.

As used herein, the expression “linked to a cell binder” refers to a conjugate molecule comprising one or more CC-1065 analog prodrugs bound to the cell binder via a suitable linker or precursor thereof; Preferred linkers contain thiols, sulfide or disulfide bonds, or precursors thereof.

The expression “cell binding agent” herein also includes modified cell binding agents, such cell binding agents for the binding of reactive cell groups such as the mercapto, sulfide or disulfide bonds of linkers of CC-1065 analogue prodrugs. Modified by modifiers that improve reactivity. Such modifiers include N-sulfosuccinimidyl-4- (5-nitro-2-pyridyldithio) butanoate (SSNPB), succinimidyl 4- [N-maleimidomethyl] cyclohexane-1- Carboxylate (SMCC), 4- (2-pyridyldithio) butanoic acid N-hydroxysuccinimide ester (SPDB), and other agents as discussed below.

As used herein, the term “analogue” refers to a compound that includes a chemically modified form of a specific compound or class thereof and retains the characteristic pharmaceutical and / or pharmacological activity of that compound or class.

As used herein, the term “patient” refers to an animal suffering from, or likely to suffer from, one or more of the diseases and disorders described herein, eg, an animal useful for breeding, business, or protective purposes, or preferably Refers to human or child.

As used herein, “therapeutically effective amount” refers to an amount of a compound of the invention that is effective for preventing, decreasing, eliminating, treating or inhibiting the diseases and disease symptoms described herein. The term “inhibition” refers to any process that can slow, block, arrest or stop the progression of the diseases and conditions described herein, but does not necessarily mean the complete elimination of all diseases and disease symptoms, Prophylactic treatment is included.

As used herein, the term “pharmaceutically acceptable” means human and animal to the extent appropriate for reasonable benefit / hazardous ratios without causing excessive toxicity, irritation, allergic reactions or other problematic complications, within the scope of careful medical judgment. It refers to a compound, substance, excipient, composition or dosage form suitable for contact with tissues of.

As used herein, “pharmaceutically acceptable salts” refers to derivatives of such compounds, which are modified by making acid or base salts of the parent compound. Pharmaceutically acceptable salts include, for example, conventional nontoxic salts or quaternary ammonium salts of the parent compound formed from nontoxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; And organic acids such as acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucononic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, maleic acid, lactic acid, and the like. Salts are included. Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine and the like, metal salts such as sodium, potassium, calcium, zinc or magnesium.

Pharmaceutically acceptable salts of the invention can be synthesized by conventional chemical methods from the parent compound containing a basic or acidic moiety. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. For a list of suitable salts, see the following references incorporated herein by reference: Remington's Pharmaceutical Sciences , 17 ^th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418].

According to a further object, the present invention also relates to a process for preparing the compound of the present invention.

The compounds and processes of the present invention can be prepared in a number of ways well known to those skilled in the art. The compounds of the present invention can be synthesized, for example, by applying or adapting the methods described below, or by making variations as is well recognized by those skilled in the art. Appropriate modifications and substitutions are readily apparent and well known or can be readily obtained from scientific journals of ordinary skill in the art.

In particular, this method may be referred to the following literature [RC Larock, Comprehensive Organic Transformations , Wiley-VCH Publishers, 1999].

It will be appreciated that the compounds of the present invention may contain one or more asymmetrically substituted carbon atoms and may be separated in optically active or racemic forms. Thus, unless specific stereochemical or isomeric forms are specifically indicated, all chiral, diastereomeric, racemic and all geometric isomeric forms of a particular structure are contemplated. Methods of making and isolating such optically active forms are well known in the art. For example, mixtures of stereoisomers can be prepared using standard techniques such as degradation of racemic forms, normal, reversed and chiral chromatography, preferential salt formation, recrystallization, and the like; Or by chiral synthesis from chiral starting materials or by deliberate synthesis of the target chiral center.

The compounds of the present invention can be prepared by various synthetic routes. Reagents and starting materials are commercially available or can be readily synthesized by those skilled in the art by well known techniques. Unless otherwise indicated, all substituents are as defined above.

In the reactions described below, it is necessary to protect reactive functional groups such as hydroxy, amino, imino, thio or carboxy groups, which are desired to be preserved in the final product to avoid unnecessary participation in the reaction. Conventional protecting groups can be used according to standard practice. See, eg, TW Greene and PGM Wuts in Protective Groups in Organic Chemistry , 3 ^rd ed., John Wiley and Sons, 1999; JFW McOmie in Protective Groups in Organic Chemistry , Plenum Press, 1973.

Some reactions may be carried out in the presence of a base. There is no particular limitation as to the type of base to be used in this reaction, and any base commonly used in this type of reaction can likewise be used herein, provided that it does not adversely affect other parts of the molecule. Examples of suitable bases include sodium hydroxide, potassium carbonate, triethylamine, alkali metal hydrides such as sodium hydride and potassium hydride; Alkyllithium compounds such as methyllithium and butyllithium; And alkali metal alkoxides such as sodium methoxide and sodium ethoxide.

Typically, the reaction can be carried out in a suitable solvent. Various solvents may be used provided that they do not adversely affect the reagents involved or the reaction itself. Examples of suitable solvents include hydrocarbons which may be aromatic, aliphatic or cycloaliphatic hydrocarbons, for example hexane, cyclohexane, benzene, toluene and xylene; Amides such as dimethylformamide; Alcohols such as ethanol and methanol; And ethers such as diethyl ether and tetrahydrofuran.

The reaction can be carried out over a wide temperature range. In general, the inventors have found it convenient to carry out the reaction under 0 to 150 ° C (more preferably about room temperature to 100 ° C). The time required for the reaction may vary greatly depending on many factors, especially the reaction temperature and the type of reagent. However, if the reaction is carried out under the above-mentioned preferred conditions, 3 to 20 hours will usually be sufficient.

The compound thus prepared can be recovered from the reaction mixture by conventional means. For example, after distilling off the solvent from the reaction mixture, or distilling off the solvent from the reaction mixture, if necessary, the residue is poured into water and then extracted with a water-immiscible organic solvent, and the solvent is distilled off from this extract. The compound can be recovered by doing so. In addition, the product may be further purified, if desired, by various well-known techniques, for example recrystallization, reprecipitation or various chromatographic techniques, in particular column chromatography or preparative thin layer chromatography.

A further object of the invention is a process for the preparation of compounds of the formula according to the invention.

The process for the preparation of the compounds of formula according to the invention comprises the step of protecting the phenolic groups of the alkylated portion of the corresponding analog of CC-1065 by sulfonic acid containing carbamate functional groups. This reaction can be carried out by applying or adapting the method of protecting an OH to a carbamate functional group, as described in the following described in reference:. TW Greene and PGM Wuts in Protective Groups in Organic Chemistry, 3 rd ed, John Wiley and Sons, 1999 or JFW McOmie in Protective Groups in Organic Chemistry , Plenum Press, 1973.

In general, the reaction is carried out using CDI / DMA and the corresponding aminophenylsulfonate derivatives of the formula:

In the above formula,

R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are as defined in formula (XII).

The process may comprise an additional step of separating the product obtained.

Preparation of Cell Binding Agents

The prodrug compounds of the invention can be used as conjugates with cell binding agents as effective therapeutic agents. Cell binding agents may be of any kind known or to be known now, including peptides and non-peptides. Generally, they are antibodies (especially monoclonal antibodies), or antibody fragments containing one or more binding sites, lymphokines, hormones, growth factors, nutrient-transporting molecules (eg transferrin), or any other cell binding agent. It may be a molecule or a substance.

More specific examples of cell binding agents that may be used include the following:

Monoclonal antibodies;

Single chain antibodies;

Fragments of the antibodies, eg Fab, Fab ', F (ab') ₂ and Fv [see; Parham, 131 J. Immunol. 2895-2902 (1983); Spring et al, 113 J. Immunol. 470-478 (1974); Nisonoff et al, 89 Arch. Biochem. Biophys. 230-244 (1960);

Interferon;

Peptides;

Lymphokines, for example IL-2, IL-3, IL-4, IL-6;

Hormones such as insulin, TRH (tyrotropin releasing hormone), MSH (melanin cell-stimulating hormone), steroid hormones such as androgens and estrogens;

Growth factors and colony stimulating factors such as EGF, TGFα, insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF and GM-CSF [Burgess, 5 Immunology Today 155- 158 (1984);

Vitamins, for example folate; And

Transferrin [see O'Keefe et al, 260 J. Biol. Chem. 932-937 (1985).

Monoclonal antibody technology allows the production of highly selective cell binding agents in the form of specific monoclonal antibodies. Particularly well known in the art are mice, rats, hamsters or any other mammals of interest, eg, original target cells, antigens isolated from target cells, complete viruses, attenuated complete viruses, and viral proteins. For example monoclonal antibodies produced by immunization with viral envelope proteins.

Selecting the appropriate cell binding agent is selected according to the particular cell population to be targeted, but in general monoclonal antibodies are preferably available where appropriate.

For example, the monoclonal antibody MY9 is a murine IgG ₁ antibody that specifically binds to the CD33 antigen (JD Griffin et al 8 Leukemia Res., 521 (1984)), wherein the target cell is acute myeloid leukemia (AML). ) Can be used to express CD33 as in disease. Similarly, monoclonal antibody anti-B4 is a murine IgG ₁ that binds to CD19 antigen on B cells and is described by Nadler et al, 131 J. Immunol. 244-250 (1983)], which can be used when the target cell is a B cell or diseased cell that expresses the antigen, such as in non-Hodgkin's lymphoma or chronic lymphocytic leukemia.

Additionally, GM-CSF which binds to bone marrow cells can be used as cell binding agent for diseased cells from acute myeloid leukemia. IL-2, which binds to activated T-cells, can be used to prevent graft rejection, to treat and prevent graft-versus-host disease, and to treat acute T-cell leukemia. MSH that binds to melanocytes can be used to treat melanoma.

Preparation of Prodrug Conjugates

Conjugates of prodrugs and cell binders can be formed using any technique. Indolyl, benzofuranyl, bis-indolyl, bis-benzofuranyl, indolyl-benzofuranyl or benzofuranyl-indolyl derivatives coupled with seco- CBI analogues are prepared to contain free amino groups, The acid labile linker or light labile linker may be used to link the antibody or other cell binder. Prodrug compounds can be condensed with peptides with suitable sequences and then linked with cell binders to generate peptidase labile linkers. Cytotoxic compounds containing primary hydroxyl groups are prepared and succinylated and linked to cell binders to generate conjugates, which can be cleaved by intracellular esterases to release free prodrugs. Preferably, synthesis of prodrug compounds containing free or protected thiol groups, with or without PEG-containing spacers, is followed by one or more sulfide, disulfide or thiol-containing prodrugs to be disulfide bonds or thioether bonds. It is easily covalently linked to the cell binding agent through.

Representative conjugates of the invention include prodrugs and antibodies, antibody fragments, epidermal growth factor (EGF), melanocyte stimulating hormone (MSH), thyroid stimulating hormone (TSH), estrogens, estrogen analogs, androgens and androgen analogs of CC-1065 analogs. It is a conjugate with.

Representative examples of making various conjugates of prodrugs of the CC-1065 analog and cell binders are described below.

Disulfide linker : anti-huMy-9-6 is the genetic of the murine monoclonal antibody My-9-6 induced against the CD33 antigen found on the surface of human bone marrow cells, including the majority of acute myeloid leukemia (AML) cases Humanized forms [see: EJ Favaloro, KF Bradstock, A. Kabral, P. Grimsley & MC Berndt, Disease Markers , 5 (4): 215 (1987); MG Hoffee, D. Tavares, RJ Lutz, Robert J., PCT Int. Appl. (2004) WO 20043344. My-9-6 can be used to prepare conjugates. This antibody was previously reported as described in J. Carlsson, H. Drevin & R. Axen, Biochem. J. , 173: 723 (1978)] is modified with N-succinimidyl-3-pyridyldithio propionate to introduce an average of four pyridyldithio groups per antibody molecule. The antibody thus modified is reacted with a thiol containing prodrug to produce a disulfide linked conjugate.

Thioether Linkers : Thiol-containing derivatives of the present invention can be linked to antibodies and other cell binders via thioether linkers as previously reported [US Pat. No. 5,208,020]. Such antibodies or other cell binding agents are commercially available compounds such as N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclo Hexane-1-carboxy- (6-amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC), can be modified. These crosslinkable reagents form non-cleavable linkers derived from maleimido-based residues.

Crosslinking reagents comprising haloacetyl-based residues include N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimi Dill bromoacetate (SBA) and N-succinimidyl 3- (bromo-acetamido) propionate (SBAP). These crosslinkable reagents form non-cleavable linkers derived from haloacetyl-based residues. Modified cell binders can be reacted with thiol containing drugs to provide thioether-linked conjugates.

Acid labile linkers : The amino group containing prodrugs of the present invention are reported as previously described by WA Blattler et al, Biochemistry 24, 1517-1524 (1985); US Pat. Nos. 4,542,225, 4,569,789, 4,618,492, 4,764,368] can be linked to antibodies and other cell binding agents through acid labile linkers.

Similarly, the hydrazido group-containing prodrugs of the invention can be linked to the carbohydrate moiety and other cell binders of an antibody via an acid labile hydrazone linker. Examples of hydrazone linkers are described in BC Laguzza et al, J. Med. Chem., 32, 548-555 (1989); RS Greenfield et al, Cancer Res., 50, 6600-6607 (1990)).

Light labile linkers : The amine group containing prodrugs of the present invention have been previously described as described in P. Senter et al, Photochemistry and Photobiology , 42, 231-237 (1985); US Patent No. 4,625,014] can be linked to antibodies and other cell binders via a light labile linker.

Peptidase labile linkers : The amine group containing prodrugs of the invention can also be linked to cell binders via peptide spacers. Short peptide spacers between drugs and macromolecular protein carriers have been previously found to be stable in serum but readily hydrolyzed by intracellular peptidase [see A. Trouet et al, Proc. Natl. Acad. Sci., 79, 626-629 (1982). The amino group containing prodrug is condensed with the peptide using a condensing agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide-HCl (EDC-HCl) to obtain a peptide derivative, which is added to the cell binder. Can be connected.

Esterase labile linkers : The prodrugs of the present invention having hydroxy alkyl groups can be succinylated with succinic anhydride and then linked to cell binders to generate conjugates, which are incorporated into intracellular esterases. Can be cleaved to release free drug (see, eg, E. Aboud-Pirak et al, Biochem Pharmacol ., 38, 641-648 (1989)).

CC-1065 analogue conjugates of antibodies, antibody fragments, protein or peptide hormones, protein or peptide growth factors and other proteins are prepared in the same manner by known methods. For example, peptides and antibodies include N-succinimidyl 3- (2-pyridyldithio) propionate, N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP), 4 -Succinimidyl-oxycarbonyl-α-methyl-α- (2-pyridyldithio) -toluene (SMPT), N-succinimidyl-3- (2-pyridyldithio) butyrate (SDPB), Succinimidyl pyridyl-dithiopropionate (SPDP), 4- (2-pyridyldithio) butanoic acid N-hydrosuccinimide ester (SPDB), succinimidyl 4- [N-maleimidomethyl] Cyclohexane-1-carboxylate (SMCC), N-sulfosuccinimidyl-3- (2- (5-nitro-pyridyldithio) butyrate (SSNPB), 2-iminothiolane or S-acetylsuccinic acid Crosslinking reagents, such as anhydrides, can be modified by known methods. See Carlsson et al, 173 Biochem. J. 723-737 (1978); Blattler et al, 24 Biochem . 1517-1524 (1985). Lambert et al, 22 Biochem . 3913-3920 (1983); Klotz et al, 96 Arch. Biochem. Biophy . s 605 (1962);. and Liu et al, 18 Biochem 690 (1979), Blakey and Thorpe, 1 Antibody, Immunoconjugates & Radiopharmaceuticals, 1-16 (1988), Worrell et al 1 Anti-Cancer Drug Design 179-184 (1986)] The free or protected thiol containing cell binder thus obtained is then reacted with a disulfide- or thiol-containing CC-1065 prodrug analog to generate a conjugate, which is prepared by standard column chromatography, HPLC or gel. It can be purified by filtration.

Preferably, the monoclonal antibody- or cell binder-CC-1065 prodrug analog conjugate is one that is linked via disulfide bonds, as discussed above, capable of delivering a CC-1065 prodrug analog.

Such cell binding conjugates can be prepared by known methods, for example by modifying monoclonal antibodies with succinimidyl pyridyl-dithiopropionate (SPDP). Carlsson et al, 173 Biochem. J. 723-737 (1978). The resulting thiopyridyl group is then transposed by treatment with a thiol containing CC-1065 prodrug analog to produce a disulfide linked conjugate. Conjugation by this method is described in detail in US Pat. No. 5,585,499, which is incorporated herein by reference.

On the other hand, in the case of aryldithio-CC-1065 prodrug analogues, cell-bonded conjugates are formed by directly transposing the aryl-thiol of the CC-1065 prodrug analogues with sulfhydryl groups previously introduced into the antibody molecule. . Conjugates containing 1 to 10 CC-1065 prodrug analogs linked through disulfide bridges are readily prepared by either method.

According to a further object, the present invention also relates to a pharmaceutical composition comprising a conjugate molecule of the invention or a compound of formula (I) as defined above together with a pharmaceutically acceptable carrier.

According to a further object, the present invention also provides for inhibiting or killing the growth of cells, preferably selected cell populations, comprising contacting the target cell or tissue containing the target cell with an effective amount of the pharmaceutical composition according to the invention. It is about a method.

The cell population of choice is cancerous and / or proliferative cells.

According to a further object, the present invention also relates to a method for treating cancer, preferably a method for selectively treating cancer, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition according to the invention. .

According to the present invention, "selective treatment of cancer" refers to killing cancerous cells and / or proliferative cells without substantially killing normal and / or non-proliferative cells.

According to a further object, the present invention also relates to the use of a conjugate molecule of the invention or a compound of formula (I) as defined above for the manufacture of a medicament for the treatment of cancer.

Methods for inhibiting the growth of selected cell populations can be performed in vitro, in vivo or ex vivo.

Examples of in vitro use include treating cell culture to kill all cells except for the desired variant that does not express the target antigen or to kill variants that express undesirable antigens.

Non-clinical in vitro use conditions can be readily determined by one skilled in the art.

Examples of ex vivo use include treating such bone marrow prior to transplanting its own bone marrow into the same patient to kill the diseased or malignant cells; Treatment of prior bone marrow transplantation to kill qualified T cells and prevent graft-versus-host disease (GVHD).

To remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or autoimmune disease treatment, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplantation to prevent GVHD Clinical ex vivo treatment can be performed as follows. Bone marrow is harvested from patients or other individuals, and then incubated in a medium containing serum to which the cytotoxic agent of the invention is added at a concentration ranging from about 10 μM to 1 pM at about 37 ° C. for about 30 minutes to about 48 hours. do. The exact incubation time and concentration conditions (= dose) can be readily determined by one skilled in the art. After incubation, the bone marrow cells were returned to the patient by washing with serum containing medium and intravenously injecting according to known methods. Under the circumstances of providing the patient with other treatments, such as systemic irradiation or ablation chemotherapy, between the time of bone marrow harvest and the time of reinjecting the treated cells, the treated bone marrow cells may be removed using standard medical equipment. Store frozen in nitrogen.

For clinical in vivo use, the cytotoxic agents of the present invention may be supplied as a solution to test for sterile and endotoxin levels, or may be supplied as lyophilized solids and redissolved in sterile water for injection. Examples of suitable protocols for conjugate administration are as follows: The conjugate is administered by intravenous bolus weekly for 6 weeks. The bolus dose is given in 50 to 400 ml of normal saline to which human serum albumin can be added (e.g., 0.5 to 1 ml concentrated human serum albumin solution, 100 mg / ml). The dosage will be about 50 μg to 10 mg / kg body weight (intravenously) per week (10 μg to 100 mg / kg per injection). Six weeks after treatment, the patient may undergo a second course of treatment. Specific clinical protocols with respect to the route of administration, excipients, diluents, dosages, times, and the like can be determined by one skilled in the art based on clinical context.

Examples of medical conditions that can be treated according to in vivo or ex vivo methods of killing selected cell populations include all types of malignant tumors, such as lung cancer, breast cancer, colon cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer and lymphatic organs. cancer; Melanoma; Autoimmune diseases such as systemic lupus, rheumatoid arthritis and multiple sclerosis; Graft rejection, eg kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection and bone marrow transplant rejection; Graft versus host disease; Viral infections such as CMV infection, HIV infection, AIDS, and the like; Bacterial infections; And parasitic infections such as flagella, amoeba, schistosomiasis, and other diseases as determined by one skilled in the art.

Identification of the subjects in need of treatment of the diseases and disorders described herein is within the skill and knowledge of those skilled in the art. Veterinarians or specialists in the art can readily identify subjects in need of such treatment by using clinical trials, physical examinations, medical / family history or biological and diagnostic tests.

The therapeutically effective amount can be readily determined by the attending physician by using conventional techniques and observing the results obtained under similar circumstances. In determining the therapeutically effective amount, a number of factors are considered by the attending physician, including the species of the subject; Its size, age and general state of health; Related specific diseases; Prevalence or disease severity; The response of the individual subject; The particular compound administered; Mode of administration; Bioavailability characteristics of the administered agents; Selected dose regimen; Whether or not to use co-administered drugs; And other related situations, including but not limited to.

The amount of compound or conjugate of formula (I) required to achieve the desired biological effect depends on a number of factors, for example the chemical characteristics (eg hydrophobicity) of the compound used, the potency of the compound, the type of disease, Species present, disease state of the patient, route of administration, efficiency of bioavailability of the compound by the selected route; It will vary depending on all factors that dictate the dosage, delivery and therapy required to administer.

As used herein, “pharmaceutically acceptable excipients” include any carrier, diluent, adjuvant or vehicle, such as preservatives or antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersions Media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of agents and media for such pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations.

In the context of the present invention, “treating” or “treatment” as used herein refers to reversing, alleviating, or reducing the disorder or condition to which such term applies, or one or more symptoms of such disorder or disease. To inhibit or prevent progression.

In a general sense, the compounds of the present invention may be provided in aqueous physiological buffer solutions containing 0.1 to 10% w / v compounds for parenteral administration. A typical dosage range is 1 μg / kg body weight to 0.1 g / kg body weight per day; Preferred dosage ranges are 0.01 mg / kg body weight to 10 mg / kg body weight or equivalent dose per day in human children. Preferred dosages of the drug to be administered are the extent and type of disease or disorder progressed, the general health of the particular patient, the relative biological efficacy of the selected compound, the formulation of the compound, the route of administration (intravenous, intramuscular, etc.), the chosen route of delivery. It is expected to depend on the pharmacokinetic properties of the compound by and variables such as the rate of administration (temporary or continuous infusion) and the schedule of administration (number of half-times for a given time).

The compounds of the present invention may also be administered in unit dosage form, wherein the term “unit dosage” may be administered to a patient and may be easily manipulated and packaged such that the physical and chemically stable units comprise the active compound itself. By a single dose is meant as a dose or as a pharmaceutically acceptable composition as described below. Thus, a typical daily total dose range is 0.01 to 100 mg / kg body weight. According to general guidelines, in humans the unit dose ranges from 1 mg to 3000 mg per day. Preferably, the unit dose ranges from 1 to 500 mg, which is administered once to six times daily, even more preferably from 10 mg to 500 mg once daily. The compounds provided herein can be formulated into pharmaceutical compositions by mixing them with one or more pharmaceutically acceptable excipients. Such unit dose compositions are for oral administration, in particular in the form of tablets, simple capsules or soft gel capsules; Or for intranasal administration, in particular in the form of powders, nasal drops or aerosols; Or for intradermal administration, eg, typically in the form of ointments, creams, lotions, gels or sprays, or via transdermal patches.

Such compositions may be convenient to administer in unit dosage form, which may be prepared by methods well known in the art of pharmacy, for example, as described in Remington: The Science and Practice of Pharmacy. , 20 ^th ed .; Gennaro, AR, Ed .; Lippincott Williams & Wilkins: Philadelphia, PA, 2000].

Preferred formulations include pharmaceutical compositions wherein the compounds of the invention are formulated for oral or parenteral administration.

For oral administration, tablets, pills, powders, capsules, troches and the like may contain one or more of the following ingredients, or compounds of a similar class: binders, for example microcrystalline cellulose or tragacanth gum ; Diluents such as starch or lactose; Disintegrants such as starch and cellulose derivatives; Lubricants such as magnesium stearate; Glidants such as colloidal silicon dioxide; Sweetening agents such as sucrose or saccharin; Or flavoring agents such as peppermint or methyl salicylate. Capsules may generally be in the form of soft or hard capsules made from gelatin blends optionally mixed with a plasticizer, as well as starch capsules. In addition, dosage unit forms may contain a variety of other materials that modify the physical form of such dosage units, such as sugar coatings, shellac or enteric agents. Syrups or elixirs in other oral dosage forms may contain sweetening, preservatives, dyes, colorants and flavorings. In addition, the active compounds may be incorporated into rapid dissolving, modified release or sustained release formulations and formulations, which are preferably bi-modal. Preferred tablets contain lactose, corn starch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate or talc in any combination.

Liquid preparations for parenteral administration include sterile aqueous or non-aqueous solvents, suspensions and emulsions. Liquid compositions may include binders, buffers, preservatives, chelating agents, sweetening agents, flavoring agents, coloring agents, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycols, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohol with water, buffered media, and saline. In particular, biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients in controlling the release of the active compound. Intravenous vehicles can include fluid and nutrient supplements, electrolyte supplements such as those based on Ringer dextrose, and the like. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Another mode of administration includes inhalation formulations, including means such as dry powders, aerosols or drops. It may be, for example, an aqueous solution containing polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or an oily solution for administration in the form of nasal drops, or a gel to be administered intranasally. Formulations for intranasal administration include, for example, lozenges or pastilles, which may include flavor bases such as sucrose or acacia and other excipients such as glycocholate. Formulations suitable for rectal administration are preferably presented as unit-dose suppositories with solid carriers, for example cocoa butter, which may include salicylates. Formulations suitable for topical application to the skin preferably take the form of ointments, creams, lotions, pastes, gels, sprays, aerosols or oils. Carriers that can be used include petroleum jelly, lanolin, polyethylene glycol, alcohols or combinations thereof. Formulations suitable for transdermal administration may be presented as separate patches, may be lipophilic emulsions or buffered aqueous solutions or may be dissolved and / or dispersed in a polymer or adhesive.

In vitro Cytotoxicity of Conjugates Between Prodrugs and Cell Binding Agents of the Invention

The cytotoxicity of the prodrugs of the invention, and the conjugates between them and the cell binder, can be measured after cleavage of the protecting group and conversion to the active drug. Cytotoxicity to non-adherent cell lines such as Namalwa and HL60 can be measured by de-extrapolating the cell proliferation curves as described below. Goldmacher et al, 135 J. Immunol . 3648-3651 (1985). Cytotoxicity of these compounds against adherent cell lines, such as A-375 and SCaBER, can be determined by clonality assays as described below. Goldmacher et al, 102 J. Cell Biol . 1312-1319 (1986).

Therapeutic Agents and Methods for Inhibiting Growth of Selected Cell Populations

The present invention also provides a therapeutic for inhibiting the growth of a selected cell population comprising:

(a) at least one of the aforementioned prodrugs in a cytotoxic amount associated with a cell binding agent; And

(b) pharmaceutically acceptable carriers, diluents or excipients.

Similarly, the present invention includes contacting a tissue or cell population suspected of containing cells from a selected cell population with a cytotoxic amount of cytotoxic agent comprising one or more of the aforementioned prodrugs associated with a cell binding agent. To inhibit the growth of selected cell populations.

Cytotoxic agents are prepared as mentioned above.

Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and can be determined by one skilled in the art based on clinical circumstances.

Examples of suitable carriers, diluents and / or excipients include (1) Dulbecco phosphate buffered saline containing pH of about 1 mg / ml to 25 mg / ml human serum albumin (pH about 7.4), (2) 0.9% saline (0.9% w / v NaCl) and (3) 5% (w / v) dextrose.

Methods for inhibiting the growth of selected cell populations can be performed in vitro, in vivo or ex vivo.

Examples of in vitro use include treating cell culture to kill all cells except for the desired variant that does not express the target antigen or to kill variants that express undesirable antigens.

Non-clinical in vitro use conditions can be readily determined by one skilled in the art.

Examples of ex vivo use include treating such bone marrow prior to transplanting its own bone marrow into the same patient to kill the diseased or malignant cells; Treatment of prior bone marrow transplantation to kill qualified T cells and prevent graft-versus-host disease (GVHD).

To remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or autoimmune disease treatment, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplantation to prevent GVHD Clinical ex vivo treatment can be performed as follows. Bone marrow is harvested from patients or other individuals, and then incubated in a medium containing serum to which the cytotoxic agent of the invention is added at a concentration ranging from about 10 μM to 1 pM at about 37 ° C. for about 30 minutes to about 48 hours. do. The exact incubation time and concentration conditions (= dose) can be readily determined by one skilled in the art. After incubation, the bone marrow cells were returned to the patient by washing with serum containing medium and intravenously injecting according to known methods. Under the circumstances of providing the patient with other treatments, such as systemic irradiation or ablation chemotherapy, between the time of bone marrow harvest and the time of reinjecting the treated cells, the treated bone marrow cells may be removed using standard medical equipment. Store frozen in nitrogen.

For clinical in vivo use, the cytotoxic agents of the present invention may be supplied as a solution to test for sterile and endotoxin levels, or may be supplied as lyophilized solids and redissolved in sterile water for injection. Examples of suitable protocols for conjugate administration are as follows: The conjugate is injected intravenously, weekly, for 6 weeks. The bolus dose is given in 50 to 400 ml of normal saline to which human serum albumin can be added (e.g., 0.5 to 1 ml concentrated human serum albumin solution, 100 mg / ml). The dosage will be about 50 μg to 10 mg / kg body weight (intravenously) per week (10 μg to 100 mg / kg per injection). Six weeks after treatment, the patient undergoes a second course of treatment. Specific clinical protocols with respect to the route of administration, excipients, diluents, dosages, times, and the like can be determined by one skilled in the art based on clinical context.

Examples of medical conditions that can be treated according to in vivo or ex vivo methods of killing selected cell populations include all types of malignant tumors, such as lung cancer, breast cancer, colon cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer and lymphatic organs. cancer; Melanoma; Autoimmune diseases such as systemic lupus, rheumatoid arthritis and multiple sclerosis; Graft rejection, eg kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection and bone marrow transplant rejection; Graft versus host disease; Viral infections such as CMV infection, HIV infection, AIDS, and the like; Bacterial infections; And parasitic infections such as flagella, amoeba, schistosomiasis, and other diseases as determined by one skilled in the art.

The invention will be illustrated next with reference to non-limiting examples. Unless stated otherwise, all percentages, ratios, parts, etc., are by weight.

Materials and methods

Melting points were measured using a heat transfer device and were not corrected. NMR spectra were recorded on a Bruker AVANCE400 (400 MHz) spectrometer. Chemical shifts are reported in ppm based on TMS as an internal standard. Mass spectra were acquired using a Bruker Esquire 3000 system. Ultraviolet spectra were recorded on a Hitachi U1200 spectrophotometer. HPLC was performed using a Beckman Coulter GOLD 125 system equipped with a Beckman Coulter system GOLD 168 variable wavelength detector and a Waters Radialpak (reverse phase C-18 column). Thin layer chromatography was performed on an Analtech GF silica gel TLC plate. Silica gel for use in flash column chromatography was supplied by Baker. Tetrahydrofuran was dried by distillation over sodium metal. Dimethylacetamide and dimethylformamide were dried by distillation on calcium hydride under reduced pressure. All other solvents used were reagent grade or HPLC grade.

The synthesis of prodrugs DC07 ( 7a ), DC08 (8a) and DC107 ( FIG. 4 ) is described herein. DC07 and DC08 as well as DC107 are derived from parent drugs DC1 and DC101, while DC09 (9a) can be prepared from PEGylated parent drug DC5 described in US Pat. No. 6,756,397 B2 (FIG. 3). Prodrug DC07 is extremely stable in aqueous solutions up to pH 5.5 and can be converted to parent drug DC1 by incubation in serum or plasma. These drugs also enhanced water solubility compared to DC1. When incubated DC07SMe in mouse serum, it was converted to the parent drug DC1SMe.

Human cancer cell lines HL60, Namalwa, A-375, COLO205 and Ramos were obtained from the American Type Culture Collection (ATCC). Kara is a murine tumor cell line stably transfected with human CD33 antigen.

Example 1

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonyl-phenyl) aminocarbonyloxy] -3H-benz (e) indole -3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-methyldithiopropanoyl) -amino] -1 H-indole-2-carboxamide (DCO7-SMe, 7b Manufacturing

11.6 mg (0.017 mmol) of DC1SMe (1- [S]-(chloromethyl) -5-hydroxy-3-{{5- [5- (3-methyldithiopropionyl) indole- in 2.5 ml of DMA 3.0 μl of DIPEA was added to a 2-yl-carbonylamino] indol-2-yl} carbonyl} -1,2-dihydro-3H-benz [e] indole) solution. After stirring for 3 minutes under Ar, 4.0 mg of carbonyldiimidazole (1.46 equiv) were added. The mixture was stirred for 2 hours and TLC showed that all DC1SMe had been consumed, after which freshly prepared 16 mg (3 equiv) of 3-aminobenzenesulfonate diisopropylethylamine salt (pH 5) was added. Was added. The mixture was stirred under Ar overnight and HPLC showed the reaction was complete. The mixture was evaporated with an oil pump and purified using a C-18 column (1.0 × 5 cm) eluted with 100% 0.01% acetic acid to 50% 0.01% acetic acid and 50% THF. Fractions were combined and evaporated to give 11.8 mg of the title compound (DC07SMe); ¹ H NMR (DMF-d7) 11.85 (br, 2H), 11.79 (s, 1H), 11.70 (s, 1H), 10.53 (s, 1H), 10.27 (s, 1H), 10.03 (s, 1H), 8.50 (s, 1H), 8.41 (s, 1H), 8.21 (d, 1H, J = 1.7 Hz), 8.17 (d, 1H, J = 8.4 Hz), 8.12 (m, 1H), 7.46 (dd, 1H , J = 1.9, 8.8 Hz), 7.66 (m, 2H), 7.61 (d, 1H, J = 8.9 Hz), 7.58 (m, 2H), 7.48 (d, 1H, J = 1.4 Hz), 7.45 (dd , 1H, J = 1.9, 8.8 Hz), 7.35 (d, 1H, J = 1.6 Hz), 5.01 (t, 1H, J = 10.0 Hz), 4.84 (dd, 1H, J = 2.2, 10.9 Hz), 4.49 (m, 2H), 4.21 (dd, 1H, J = 3.2, 11.3 Hz), 4.10 (m, 2H), 3.12 (t, 2H, J = 7.0 Hz), 2.49 (s, 3H); ¹³ C NMR 172.52, 170.33, 169.54, 162.94, 161.20, 160.41, 152.67, 147.94, 142.69, 139.11, 134.73, 134.56, 133.72, 133.37, 133.18, 132.16, 130.72, 128.76, 128.41, 128.125, 125.16, 125.16. , 121.51, 120.05, 118.36, 113.54, 112.97, 112.92, 112.16, 111.61, 108.14, 107.56, 106.78, 104.02, 69.03, 67.95, 67.37, 55.84, 54.91, 48.13, 42.71, 37.05, 34.10, 23.12; MS m / z ⁺ 831.14 (M + H) ⁺ , 883.0, 883.9, 884.9 (M + Na) ⁺ , 905.0, (M + K) ⁺ 920.9, 922.9; MS m / z ^- 880.8, 881.8, 882.8, 883.8 (MH) ^- .

Example 2

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylphenyl) aminocarbonyloxy] -3H-benz (e) indole- Preparation of 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide (DCO7, 7a)

A 10 mg (0.104 mmol) TCEP solution in 2 ml H ₂ O was adjusted to pH 7.0 with the addition of NaHCO ₃ powder. To this solution was added 15 mg of DC07SMe in 3 ml of DMA followed by 5 mg of DTT in 1.0 ml 20 mM NaH 2 PO 4, pH 4.5. After stirring for 2 hours and MS showed the DC07SMe peak (m / z 883 (M-1)) disappeared, the mixture was concentrated and eluted for 40 minutes with 60% of 10 mM NaH 2 PO 4 and 40% DMA to 80% DMA. Purified using preparative HPLC c-18 column. The collected fractions were combined, concentrated, redissolved in DMA, the salts were filtered off and evaporated again to give 13 mg of DC07. MS m / z ^- 835.1 (MH), 836.1, 837.1, 838.1.

Example 3

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-{[3- (2-pyridyldithio) -propanoyl] -amino} -1H-indole-2-carboxamide ( Manufacture of DC08SPy)

21.0 mg (DC1SPy) in 2 ml of DMA was added to 10 microliters of diisopropylethylamine (DIPEA). After stirring for 3 minutes under Ar, 8.0 mg of carbonyl dimidazole (CDI) was added, the mixture was stirred for 2 hours, and examined by HPLC on a C-18 column, where DC1SPy reacted completely with CDI. To this mixture was added 20 mg of 4-aminobenzyl sulfonic acid and 10 microliters of DIPEA. After stirring overnight, the mixture was concentrated and purified by HPLC on a C-18 column eluted with 10 mM NaH 2 PO 4, 30% DMA in pH 4.0 to 80 mM DMA in 10 mM NaH 2 PO 4, pH 4.0. Fractions were combined and concentrated to give 20.2 mg of the title compound. ¹ H NMR (DMF-d7) 11.67 (d, 2H, J = 9.9 Hz), 10.58 (s, 1H), 10.23 (s, 1H), 9.81 (s, 1H), 8.86 (dddd, 1H, J = 0.8 , 1.6, 2.5, 14.6), 8.41 (s, 1H), 8.27-8.20 (m, 4H), 8.12 (m, 1H), 7.95 (d, 1H, J = 8.2 Hz), 7.83 (m, 1H), 7.71 (dd, 1H, J = 2.0, 9.9 Hz), 7.60-7.51 (m, 4H), 7.47-7.40 (m, 4H), 7.30 (d, 1H, J = 1.6 Hz), 4.90 (t, 1H, J = 10.0 Hz), 4.75 (dd, 1H, J = 2.2, 10.9 Hz), 4.33 (m, 1H), 4.15 (dd, 1H, J = 3.2, 11.3 Hz), 4.11 (s, 2H), 3.97 ( dd, 1H, J = 7.8, 11.0 Hz), 3.29 (t, 2H, J = 6.9 Hz), 2.76 (m, 3H); ¹³ C NMR 172.35, 162.93, 161.20, 160.41, 155.77, 151.36, 147.94, 142.69, 140.14, 139.11, 134.73, 134.56, 133.82, 133.37, 132.97, 132.16, 131.49, 129.22, 128.82, 128.34. , 121.81, 120.05, 119.32, 116.43, 113.31, 112.97, 112.92, 112.16, 111.61, 108.14, 107.56, 106.83, 101.78, 67.37, 56.37, 48.13, 42.71, 37.00, 34.10; MS m / z ^- 958.34 (M-1).

4-aminobenzylsulfonic acid

Fill a 250 ml Par bottle with 4-nitrobenzyl sulfonic acid (6.80 g, 31.34 mmol), 10% Pd / C (0.4 g) and CH ₃ OH (150 ml) with a few drops of water. Put and purged with hydrogen. The reaction mixture was shaken with 20 psi H ₂ overnight. The catalyst was removed by filtration through celite and the solvent was evaporated. The crude compound was co-evaporated three times with 3 × 50 ml of water and crystallized with water / THF / EtAc / toluene (1: 10: 10: 100) to give 5.3 g (91%) of the title compound. ¹ H NMR (D 2 O), 7.28 (dd, 2H, J = 2.2, 4.3 Hz), 6.95 (dd, 2H, J = 2.2, 4.3 Hz), 4.10 (s, 2H); ¹³ C NMR 134.30, 127.10, 121.04, 120.42, 59.14; MS m / z ^- 186.35 (MH).

4-nitrophenylthiol acetate

To 5 ml (69.95 mmol) thiolacetic acid and 12 ml triethyl amine solution in 75 ml toluene was added dropwise 7.5 g (34.71 mmol) 4-nitrobenzyl bromide in 100 ml 1: 2 THF / toluene for 4 hours. It was. The mixture was continuously stirred overnight, evaporated and then crystallized with EtAc / toluene / hexanes to give 7.1 g (96%) of the title compound. ¹ H NMR (CDCl 3) 8.13 (ddd, 2H, J = 2.5, 4.5, 9.3 Hz), 7.44 (ddd, 2H, J = 2.5, 4.5, 9.3 Hz), 4.14 (s, 2H), 2.35 (S, 3H ); ¹³ C NMR 194.53, 145.73, 129.91, 124.07, 32.93, 30.50; MS M ⁺ 234.34 (M + Na).

4-nitrobenzyl sulfonic acid

7.0 g of 4-nitrophenylthiol acetate was added to 150 ml of a 1: 3 H ₂ O ₂ / HAc solution in 5 portions for 4 hours, and the mixture was continuously stirred overnight. The mixture was evaporated and co-evaporated three times with 3 x 50 ml water and then crystallized with CH3OH / THF / toluene to give 6.82 g (95%) of the title compound. ¹ H NMR (D 2 O) 8.02 (ddd, 2H, J = 2.7, 4.6, 11.1 Hz), 7.20 (ddd, 2H, J = 2.6, 4.6, 11.1 Hz), 4.11 (s, 2H); ¹³ C NMR 141.70, 139.30, 129.91, 122.07, 59.15; MS m / z ^- 216.21 (MH).

Example 4

(S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- Preparation of 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1 H-indole-2-carboxamide (DC08)

A 10 mg (0.104 mmol) TCEP solution in 2 ml H ₂ O was adjusted to pH 7.0 with the addition of NaHCO ₃ powder. To this solution was added 15 mg of DC08SPy in 3 ml of DMA followed by 5 mg of DTT in 1.0 ml 200 mM NaH 2 PO 4, pH 4.5. After stirring for 2 hours and MS showed the DC08SPy peak (m / z 883 (M-1)) disappeared, the mixture was concentrated and 60% of 10 mM NaH 2 PO 4/40% DMA to 20% of 10 mM NaH 2 PO 4/80% Purification using a preparative HPLC c-18 column eluted with DMA for 40 minutes. The collected fractions were combined, concentrated to dryness, redissolved in DMA, the salts were filtered off and evaporated again to give 13 mg of DC08. MS m / z ^- 849.1 (MH), 850.1, 851.1.

Example 5

(S) -N- (2- (1- ( Chloromethyl ) -5-hydroxy-2,3- Dehydro -1H- Benzo (e) indole -3- Carbonyl ) -1H-indol-5-yl) -3- ( Methyldisulfanyl ) Propanamide  ( DC101SMe Manufacturing

5-hydroxy-3-amino-1- [S]-(chloromethyl) -1,2-dihydro-3H-benz (e) indole, hydrochloride salt (7.0 mg, 0.57 mmol) in 7.0 ml DMA And EDC (300 mg, 1.56 mmol) was added under Ar to a solution of 5- (3- (methyl-disulfanyl) propaneamido) -1H-indole-2-carboxylic acid (170 mg, 0.55 mmol). After stirring overnight, the mixture was evaporated to dryness and purified by SiO ₂ chromatography (20% to 40% THF in toluene) and crystallized with THF / toluene / hexanes to give 218 mg (76%) of DC ₁₀₁ SMe. . ¹ H NMR (DMF-d ₇ ) 11.63 (s, 1H), 10.56 (s, 1H), 9.95 (s, 1H), 8.23 (s, 2H), 7.93 (d, 1H, J = 8.3 Hz), 7.56 (dd, 1H, J = 2.0, 8.1 Hz), 7.52 (s, 1H), 7.47 (dd, 1H, J = 1.9, 8.9 Hz), 7.41 (d, 1H, J = 1.7 Hz), 7.25 (d, 1H, J = 1.7 Hz), 4.88 (dd, 1H, J = 8.7, 11.0 Hz), 4.73 (dd, 1H, J = 2.3, 10.9 Hz), 4.31 (m, 1H), 4.12 (dd, 1H, J = 3.1, 11.0 Hz), 3.95 (dd, 1H, J = 8.4, 11.2 Hz), 3.21 (t, 2H, J = 7.1 Hz), 2.71 (t, 2H, J = 7.1 Hz), 2.45 (s, 3H ); ¹³ C NMR 169.54, 161.06, 155.36, 134.21, 133.39, 132.54, 131.05, 128.39, 127.99, 123.98, 123.74, 123.46, 123.31, 118.88, 115.99, 112.87, 112.31, 106.40, 101.33, 55.93, 48.93, 48.93, 48.93. , 25.34. MS m / z + 548.2 (M + Na), 550.2 (M + 2 + Na).

5- (3- (Methyldisulfanyl) propaneamido) -1 H-indole-2-carboxylic acid

To a solution of 3- (methyldisulfanyl) propanoic acid (1.18 g, 7.76 mmol) in 50 ml of DMA was added hydroxysuccinimide (1.14 g, 9.90 mmol) and EDC (1.91 g, 9.96 mmol) under argon. It was. The mixture was stirred overnight. After confirming that the reaction was complete by TLC (1: 6 EtAc / hexanes), the mixture was used directly in the next step without further purification.

To the solution of NHS ester prepared above was added dropwise to a solution of 5-amino-1H-indole-2-carboxylic acid (1.32 g, 7.49 mmol) in 50 ml 1: 4 0.5 M Na2HPO4, pH 8.0 / DMA at 0 ° C. for 1 hour. It was. After addition, the mixture was continuously stirred at 4 ° C. for 1 hour and then at room temperature for 1 hour. The mixture was concentrated and purified on SiO2 chromatography eluted with THF / toluene / acetic acid (1: 4: 02% to 1: 2: 0.02%) to give the title product (1.62 g, 70%). 1 H NMR (DMF-d7) 12.80 (br, 0.8H), 11.66 (s, 1H), 9.96 (s, 1H), 8.17 (s, 1H), 7.44 (s, 2H), 7.14 (d, 1H, J = 2.0 Hz), 3.20 (t, 2H, J = 6.9 Hz), 2.72 (t, 2H, J = 7.0 Hz), 2.37 (s, 3H). 13C NMR 169.53, 163.45, 135.24, 133.38, 130.12, 127.99, 119.13, 113.10, 112.27, 108.04, 36.88, 30.60. MS m / z ^- 309.20 (M-1).

Example 6

(S) -3-((1- (chloromethyl) -3- (5- (3- (methyldisulfanyl) propaneamido) -1H-indole-2-carbonyl) -2,3- Dehydro -1H- Benzo [e] indole -5- Iloxy ) Carbonylamino ) Benzene sulfonic acid  ( DC107SMe )

DC101SMe ((S) -N- (2- (1- (chloromethyl) -5-hydroxy-2,3-dihydro-1H-benzo [e] indole-3-carbonyl)-in 10 ml of DMA To a solution of 1H-indol-5-yl) -3- (methyldisulfanyl) propanamide, 100 mg 0.19 mmol) was added DIPEA (35 μl, 0.20 mmol). After stirring for 3 minutes under Ar, 32.0 mg (0.20 mmol) of carbonyldiimidazole were added. When the mixture was stirred for 2 hours, TLC showed that all DC101SMe were consumed, then freshly prepared 3-aminobenzenesulfonate diisopropylethylamine salt (65 mg, 0.21 mmol) was added. When the mixture was stirred under Ar for 24 hours, the HPLC completed the reaction and the reaction was complete with DC107SMe [(S) -3-((1- (chloromethyl) -3- (5- (3- (methyldisulfanyl) propaneamido ) -1H-indol-2-carbonyl) -2,3-dihydro-1H-benzo [e] indol-5-yloxy) carbonylamino) benzenesulfonic acid] was formed. This mixture was concentrated to about 5 ml using an oil pump and used directly in the next step without further purification.

Example 7

(S) -3-((1- (chloromethyl) -3- (5- (3-mercaptopropaneamido) -1H-indole-2-carbonyl) -2,3-di He Draw-1H- Benzo [e] indole -5- Iloxy ) Carbonylamino ) Benzene sulfonic acid  ( DC107 )

To the concentrated solution was added 60 mg (0.62 mmol) of TCEP in 10 ml 1: 1 DMA / 0.4 M NaH 2 PO 4, pH 4.0. After stirring for 4 h under Ar, the mixture was concentrated and purified using a C18 column eluted with 90% 0.01% acetic acid in DMA to 40% 0.01% acetic acid in DMA. Fractions were collected and evaporated to 94 mg (73%, 2 steps) of (S) -3-((1- (chloromethyl) -3- (5- (3-mercaptopropaneamido) -1H-indole-2 -Carbonyl) -2,3-dihydro-1H-benzo [e] indol-5-yloxy) carbonylamino) benzenesulfonic acid (DC107) was obtained. ¹ H NMR (DMF-d ₇ ) 11.63 (s, 1H), 10.51 (s, 1H), 9.89 (s, 1H), 8.33 (s, 2H), 7.93 (d, 1H, J = 8.3 Hz), 7.58 (m, 2H), 7.52 (m, 2H), 7.47 (m, 2H), 7.41 (m, 1H), 7.38 (m, 1H), 7.25 (d, 1H, J = 1.7 Hz), 4.87 (dd, 1H, J = 8.7, 11.0 Hz), 4.74 (dd, 1H, J = 2.3, 10.9 Hz), 4.32 (m, 1H), 4.12 (dd, 1H, J = 3.0, 11.0 Hz), 3.93 (dd, 1H , J = 8.4, 11.2 Hz), 3.21 (t, 2H, J = 7.1 Hz), 2.70 (t, 2H, J = 7.1 Hz); ¹³ C NMR 169.55, 161.30, 155.46, 150.21, 143.45, 134.22, 133.37, 132.54, 131.35, 130.31, 128.38, 127.95, 123.78, 123.72, 123.48, 123.33, 121.41, 118.88, 115.90, 112.95, 112.88, 112.95 , 48.60, 42.67, 36.90, 25.01. MS m / z-676.90 (MH), ε _{340 nm} = 19025 M ^-1 cm ^-1 .

Conjugation of Monoclonal Antibodies with DC07 . A huMy9-6 antibody that binds preferentially expressed CD33 antigen on human AML cell surface was selected for conjugation with DC07.

Example 8

huMy9-6-SSNPB-DC07: Preparation of Disulfide Linked Conjugates

In the first step, the antibody was reacted with the modifier N-sulfosuccinimidyl 5-nitro-2-pyridyldithiobutanoate (SSNPB) to introduce nitropyridyldithio groups. 7.5 times the huMy9-6 antibody solution at a concentration of 8.5 mg / mL in aqueous buffer containing 0.05 M potassium phosphate, 0.05 M sodium chloride, 2 mM ethylenediaminetetraacetic acid (EDTA), 3% dimethylacetamide (DMA), pH 6.5 Treated with molar excess of SSNPB solution (20 mM stock in DMA). The reaction mixture was stirred at room temperature for 90 minutes and purified on a Sephadex G25 pre-packed column that had previously been equilibrated into an aqueous buffer containing 0.1 M NaH ₂ PO ₄ , 50 mM NaCl, pH 6.5. The sample thus purified produced 98% of the product. Bovine aliquots of modified antibodies were treated with dithiothreitol to cleave nitro-pyridyl disulfides and assayed using a spectrophotometer on the released nitro-pyridine-2-thione: nitro-pyridine-2 - in the case of a ^{_{thione, ε 325nm = 10,964 M -1 cm}} -1 and ε _280nm = 3,344 M ^-1 cm ^-1 and, in the case of the antibody, ^{_{ε 280nm = 206,460 M -1 cm -1}} . An average of 5.0 nitro-pyridyldisulfide groups were linked per antibody molecule.

Modified antibodies were diluted to 2.0 mg / mL with 5% DMA in the above buffer at pH 6.5 and then treated with a solution of DC07 in DMA, using drug excess at 2.5 drugs per linker. The conjugation mixture was reacted for 30 minutes at room temperature. The reaction mixture was purified by passing through a Sephacryl S300 gel filtration column previously equilibrated at 20% DMA, 0.1 M NaH ₂ PO ₄ , 50 mM NaCl, pH 5.0. Fractions containing monomeric antibody-DC07 conjugates were pooled and dialyzed into 10 mM citrate, 135 mM NaCl, pH 5.0. The final conjugates were assayed spectrophotometrically using the following absorbance coefficients: for drugs, ε _{340 nm} = 38,000 M ^-1 cm ^-1 and ε _{280 nm} = 23,000 M ^-1 cm ^-1 , and for antibodies ε _{280 nm} = 206,460 M ⁻¹ cm ⁻¹ . The conjugate contained 3.5 drugs per antibody.

Example 9

huMy9-6-SMCC-DC07: Preparation of Thioether Linked Conjugates

In the first step, the antibody was reacted with the modifier succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). A solution of huMy9-6 antibody at a concentration of 8.5 mg / mL in aqueous buffer containing 0.05 M potassium phosphate, 0.05 M sodium chloride, 2 mM ethylenediaminetetraacetic acid (EDTA) and 3% DMA, pH 6.5 was 8.5-fold for 90 minutes at room temperature. Treated with molar excess of SMCC solution (20 mM in DMA). Modified antibodies were purified on Sephadex G25 pre-packed columns that had previously been equilibrated into aqueous buffer containing 0.1 M NaH ₂ PO ₄ , 50 mM NaCl, pH 6.5. The sample thus purified was assayed with a spectrophotometer (ε _{280 nm} = 206,460 M ^-1 cm ^-1 ) to calculate the concentration of the antibody. The concentration of bound linker was determined by an established procedure that indirectly measured the amount of linker available for reaction with the thiol containing amino acid cysteine. After modification, it was determined that there were 6.0 linkers per antibody.

The modified antibody was diluted to 2.0 mg / ml in 0.1 M NaH ₂ PO ₄ , 50 mM NaCl, 5% DMA, pH 6.5 and treated with 0.20 mM DC07 (2.5 mol DC07 / linker). The conjugation reaction proceeded at room temperature for 80 minutes. The reaction was then acidified with 5% acetic acid and analyzed for analysis using an HPLC system equipped with an analytical size exclusion (SEC) column equilibrated at 20% DMA, 0.1 M NaH ₂ PO ₄ , 50 mM NaCl, pH 5.0. Submitted, it was possible to isolate the antibody-drug conjugate from unreacted DC07. Eluates from the column were monitored for absorbance under 280 nM and 340 nM. The ratio of DC07 to antibody was determined by spectrophotometer to be 3.5 (for drugs, ε _{340 nm} = 38,000 M ^-1 cm ^-1 and ε _{280 nm} = 23,000 M ^-1 cm ^-1 , and for antibodies ε _280nm = 206,460 M ⁻¹ cm ⁻¹ ).

Calculation on Drug / Ab Ratio from SEC Analysis:

DC07 = A _340/38000

_{huMy9-6 = [(A 280 - (} 23000 * A 340)) / 38000] / 206460

Drug / Ab = DC07 / My9-6

Example 10

Biological results

Cytotoxicity of DC07-SMe (FIG. 2) and DC1-SMe (FIG. 1D) against Ramos and HL60 / S cells was measured with in vitro exposure for 5 days. The results are shown in FIG. IC50 is presented next.

Concentration (m) Cell line DC1-SMe DC07-SMe HL60 / s 3.6 x 10 ^-11 M 5 x 10 ^-11 M Ramos 6.2 x 10 ^-11 M 1.2 x 10 ^-10 M

Cytotoxicity of DC1-SPy and DC07-SPy against Ramos and HL60 cells was measured. The results are shown in FIG. IC50 is presented next.

DC1-SPy: DC07-SPy:

Cell Line IC ₅₀ (M) Cell Line IC ₅₀ (M)

Ramos 7.5 x 10 ^-12 Ramos 2.8 x 10 ^-11

HL60 7.5 x 10 ^-12 HL60 2.0 x 10 ^-11

Cytotoxicity of DC08-SSPy against Namalwa and Ramos cells was measured. The results are shown in FIG. IC50 is presented next.

Cell line IC ₅₀ (M) Namalwa 4.2 x 10 ^-10 M Ramos 4.2 x 10 ^-10 M

Cytotoxicity of MY9-6-DC07 (disulfide linked) against antigen positive Kara and antigen negative Ramos cells was measured. The results are shown in FIG. 10 and IC50 is shown next.

Cell line IC ₅₀ (M) Kara 8.006 x 10 ^-13 M Ramos 1.015 x 10 ^-9 M

Cytotoxicity of huC242-DC07 (thio ether linked) against antigen positive COLO 205 and antigen negative A-375 cells was measured. The results are shown in FIG. 11 and IC50 is shown next.

Cell line IC ₅₀ (M) COLO 205 3.5 x 10 ^-11 M A-375 1.3 x 10 ^-9 M

Certain patents and publications are incorporated herein by reference, the teachings of which are incorporated herein by reference in their entirety.

Although the present invention has been described in detail with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (34)

A phenolic group of the alkylated portion of the molecule is protected by a protecting group to include an analog of CC-1065 which increases the water solubility of the prodrug and further comprises a linker capable of conjugating the prodrug to the cell binder. A prodrug characterized in that the protecting group is a sulfonic acid containing carbamate. The compound of claim 1, wherein the analog of CC-1065 is a compound of Formula II, III, IV, V, VI, VII, VIII, IX, X, or XI in the secondary amino group of the pyrrole residue of the first subunit of Formula (I). Analogs formed from the first subunit covalently linked to the second subunit, or a pharmaceutically acceptable salt, hydrate or hydrate salt thereof, via an amide linkage of a second subunit to C-2 carboxyl, or Prodrugs selected from the group consisting of polymorphic crystalline structures of these compounds or optical isomers, racemates, diastereomers or enantiomers thereof: <Formula I>

<Formula II>

<Formula III>

<Formula IV>

<Formula V>

<Formula VI>

<Formula VII>

<Formula VIII>

<Formula IX>

<Formula X>

<Formula XI>

Where Y represents a leaving group selected from halides (fluoride, chloride, bromide or iodide), methanesulfonate, para-toluenesulfonate, trifluoromethane sulfonate, mononitro or dinitrophenolate; R, R ', R ₁ to R ₆ are each independently hydrogen, C ₁ -C ₃ linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino or amido, or a cell binder A linker that provides a linkage with a prodrug, provided that at least one of R, R ', R ₁ -R ₆ is a linker; R ₇ is a sulfonic acid containing carbamate protecting group. The prodrug of claim 2, wherein R ₇ is a sulfonic acid containing phenyl carbamate of formula XII: <Formula XII>

Where R ₉ is H or branched, cyclic or linear alkyl; R _10, R _11, R ₁₂ or R ₁₃ are the same or different and are one or more double -X-SO ₃ ^- groups inde ⁺ M, M ⁺ is a cation derived from the original character of the H ⁺ or a Group IA, X Is a direct bond or a spacer selected from C1 to C4 linear or branched alkylene, alkenyl or alkynyl, -Oalkyl-, -Salkyl- and aminoalkyl, and other R ₁₀ , R ₁₁ , R ₁₂ or R ₁₃ are H, C ₁ -C ₆ branched or linear alkyl, —Oalkyl, —Salkyl, hydroxyl, primary amino, secondary amino, or amido, halide, nitro, azido. 4. The prodrug of claim 3, wherein R ₉ is H. 3 according to any one of claims 4, _{wherein, R 10, R 11, R} 12, R 13 is one of -X-SO ₃ ^- and M ^+, the others are H prodrugs. The compound according to any one of claims 2 to 5, wherein R ', R _1. To a prodrug wherein R ₆ is hydrogen and R is a linker. 7. The prodrug of claim 1, wherein the linker comprises a thiol, sulfide or disulfide bond containing substituent. 8. The prodrug of claim 1, wherein the linker comprises polyethylene glycol of the formula — (OCH ₂ CH ₂ ) _n −, wherein n is an integer from 1 to 2000. 9. The prodrug of claim 1, wherein the linker group has the formula: -G-D- (Z) p-S-Z ' Where G is a single or double bond, -O-, -S- or -NT-; D is a single bond or -E-, -E-NT-, -E-NT-F-, -EO-, -EOF-, -E-NT-CO-, -E-NT-CO-F-, -E-CO-, -CO-E-, -E-CO-F, -ES-, -ESF-, -E-NT-CS-, -E-NT-CS-F-; T is H or C ₁ -C ₆ branched or linear alkyl; E and F are the same or different and are linear or branched-(OCH ₂ CH ₂ ) _i alkyl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i -alkyl-,-(OCH ₂ CH ₂ ) _i -,-(OCH ₂ CH ₂ ) _i Cycloalkyl (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i Heterocyclic (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i Aryl (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i heteroaryl (OCH ₂ CH ₂ ) _j- , -alkyl- (OCH ₂ CH ₂ ) _i alkyl (OCH ₂ CH ₂ ) _j -,- Alkyl (OCH ₂ CH ₂ ) _i- , -alkyl- (OCH ₂ CH ₂ ) _i Cycloalkyl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i Heterocyclic (OCH ₂ CH ₂ ) _j -, -Alkyl- (OCH ₂ CH ₂ ) _i aryl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i heteroaryl (OCH ₂ CH ₂ ) _j- , cycloalkyl-alkyl-, -alkyl Independently selected from -cycloalkyl-, -heterocyclic-alkyl-, -alkyl-heterocyclic-, -alkyl-aryl-, aryl-alkyl-, -alkyl-heteroaryl- and -heteroaryl-alkyl- Become; i and j are the same or different integers and are independently selected from 0, 1 to 2000; Z is linear or branched-alkyl-; p is 0 or 1; Z 'is H, a thiol protecting group, for example COR, R ₂₀ or SR _2O , where R ₂₀ represents H, methyl, alkyl, optionally substituted cycloalkyl, aryl, heteroaryl or heterocyclic. The prodrug of claim 9, wherein G is a single bond or -NT-. The prodrug of claim 9 or 10, wherein G is -NT-. The prodrug of any one of claims 9-11, wherein D is -CO-E-. 13. The prodrug of any one of claims 9-12, wherein E is linear or branched-alkyl-. The prodrug of any one of claims 9-13, wherein p is zero. 15. The prodrug of any one of claims 9-14, wherein Z 'is H or SR _2O and R ₂₀ represents alkyl or heteroaryl. The linker of claim 1, wherein the linker is (CH ₂ ) _p NHCO (CH ₂ ) _m SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m SZ, (CH ₂ ) _p. O (CH ₂ ) _m SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m (OCH ₂ CH ₂ ) _n SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n SZ , (CH ₂ ) _p O (CH ₂ ) _m (OCH ₂ CH ₂ ) _n SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p O (CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CH (Me) SZ, ( CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CH (Me) SZ, (CH ₂ ) _p O (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CH (Me) SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m C (Me) ₂ SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m C (Me) ₂ SZ, (CH ₂ ) _p O (CH ₂ ) _m C (Me) ₂ SZ, (CH ₂ ) _p NHCO (CH ₂ ) _m (OCH ₂ CH ₂ ) _n C (Me) ₂ SZ, (CH ₂ ) _p NHCOC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n C (Me) ₂ SZ, (CH ₂ ) _p O (CH ₂ ) _m (OCH ₂ CH ₂ ) _n C (Me ₂ ) SZ, (CH ₂ ) _p OC ₆ H ₄ (CH ₂ ) _m SZ , (CH ₂ ) _p OC ₆ H ₄ (CH ₂ ) _m CH (Me) SZ, (CH ₂ ) _p OC ₆ H ₄ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n CHMeSZ, (CH ₂ ) _p OC _{6 H 4 (CH 2) m} CMe 2 SZ again _{(CH 2) p OC 6 H} 4 (CH 2) m (OCH 2 CH 2) n C (Me) 2 SZ [ wherein, Z is H, a thiol protecting group, for example, represents the Ac, R ₈ or SR ₈ , R ₈ represents methyl, linear alkyl, branched alkyl, cyclic alkyl, simple or substituted aryl or heterocyclic, m represents an integer from 0 to 10, n represents an integer from 0 to 2000, p is Represents an integer of 0 to 10]. The linker of claim 1, wherein the linker is (CH ₂ ) _p NHCO (CH ₂ ) ₂ SH, (CH ₂ ) _p NHCO (CH ₂ ) ₂ C (Me) ₂ SH, (CH _2). ) _p NHCO (CH ₂ ) ₂ SSCH ₃ , (CH ₂ ) _p NHCO (CH ₂ ) ₂ (OCH ₂ CH ₂ ) _n SH, (CH ₂ ) _p NHCO (CH ₂ ) ₂ (OCH ₂ CH ₂ ) _n C A prodrug selected from (Me) ₂ SSCH ₃ and (CH ₂ ) _p NHCO (CH ₂ ) ₂ (OCH ₂ CH ₂ ) _n SSMe. 18. The compound according to any one of claims 1 to 17, wherein the compound of the formula: or a pharmaceutically acceptable salt, hydrate or hydrate salt thereof, or polymorphic crystalline structure of these compounds or optical isomers, racemates, moieties thereof Prodrugs consisting of stereoisomers or enantiomers:

Where Y, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R are as defined in any one of claims 2 to 17, and R is defined in claims 7 to 17. Is a linker as defined in any of the claims. The method according to any one of claims 1 to 18, (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-methyldithiopropanoyl) -amino] -1H-indol-2-carboxamide; (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide; (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-{[3- (2-pyridyldithio) -propanoyl] -amino} -1H-indol-2-carboxamide; (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide; (S) -N- [2-{(1-chloromethyl) -1,2-dihydro-5-[(3-hydroxysulfonylmethylphenyl) aminocarbonyloxy] -3H-benz (e) indole- 3-yl} carbonyl] -1H-indol-5-yl] -5-[(3-mercaptopropanoyl) -amino] -1H-indol-2-carboxamide; or Prodrugs which are pharmaceutically acceptable salts, hydrates or hydrated salts thereof, or polymorphic crystalline structures of these compounds or optical isomers, racemates, diastereomers or enantiomers thereof. 18. The compound according to any one of claims 1 to 17, wherein the compound of the formula: or a pharmaceutically acceptable salt, hydrate or hydrate salt thereof, or polymorphic crystalline structure of these compounds or optical isomers, racemates, moieties thereof Prodrugs consisting of stereoisomers or enantiomers:

Where Y, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R are as defined in any one of claims 2 to 17, and R is defined in claims 7 to 17. Linker as defined in any one of the preceding claims. The method according to any one of claims 1 to 17 and 20, (S) -3-((1- (chloromethyl) -3- (5- (3-mercaptopropaneamido) -1H-indole-2-carbonyl) -2,3-dihydro-1H-benzo [e] indol-5-yloxy) carbonylamino) benzenesulfonic acid; (S) -3-((1- (chloromethyl) -3- (5- (3- (methyldisulfanyl) propaneamido) -1H-indole-2-carbonyl) -2,3-dihydro -1H-benzo [e] indol-5-yloxy) carbonylamino) benzenesulfonic acid; or Prodrugs which are pharmaceutically acceptable salts, hydrates or hydrated salts thereof, or polymorphic crystalline structures of these compounds or optical isomers, racemates, diastereomers or enantiomers thereof. A prodrug conjugate comprising a cell binder linked to one or more prodrugs according to any one of claims 1 to 21 via a linking group. The prodrug conjugate of claim 22, wherein the cell binding agent is selected from antibodies and fragments thereof, interferons, lymphokines, vitamins, hormones, and growth factors. The prodrug conjugate of claim 22 or 23 wherein the cell binding agent is an antibody or fragment thereof. The prodrug conjugate of any one of claims 22-24, wherein the linking group comprises a linker linked to a functional group reactive to thiol, sulfide or disulfide. A pharmaceutical composition comprising a prodrug according to any one of claims 1 to 21 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the conjugate according to any one of claims 22 to 25 and a pharmaceutically acceptable carrier. Use of a prodrug according to any one of claims 1 to 21 for the manufacture of a medicament for the treatment of cancer. Use of a conjugate according to any one of claims 22 to 25 for the manufacture of a medicament for the treatment of cancer. 22. A process for the preparation of a compound according to any one of claims 1 to 21, comprising protecting the phenolic group of the alkylated portion of the corresponding analog of CC-1065 by a sulfonic acid containing carbamate functional group. The process of claim 30, wherein the reaction is carried out using CDI / DMA (carbonyldiimidazole / dimethylamine) and the corresponding amino-phenylsulfonate derivatives of the formula:

Where R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are as defined in claim 3. 32. The method of claim 30 or 31, further comprising hydrolyzing the product obtained. 33. The method of any one of claims 30 to 32, further comprising the step of separating the desired product. An analog of CC-1065 as defined in any one of claims 1 to 21 comprising a sulfide, disulfide or thiol group is reacted with a cell binder comprising a functional group reactive to sulfide, disulfide or thiol, 26. A method of making a conjugate according to any one of claims 22 to 25, comprising the step of connecting such analogs and cell binders to one another via covalent sulfide or disulfide bonds.

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